Process for producing an asymmetric porous film

ABSTRACT

It is intended to provide asymmetric porous films which are usable in blood dialysis, plasma separation, etc. and particularly excellent in the performance of selectively separating (fractionating) plasma protein, show little endogenous coagulation, complement or quinine activily and have an extremely high biocompatibility. Porous films made mainly of a synthetic polymer and having an asymmetric structure wherein, in the sectional structure, a dense layer substantially not charged at least on the outermost surface is provided in the side on which a liquid to be treated is loaded and at least part of the film other than the outermost surface is negatively charged. In the above films, the dense layer non-charged at least on the outermost surface serves as a size barrier while the part of the film other than the outermost surface serves as a charge barrier.

TECHNICAL FIELD

The present invention relates to an asymmetric porous membrane forseparating specific solutes and/or dispersoids from a liquid and to amethod for manufacturing the same.

The asymmetric porous membrane of the present invention exhibitsremarkably improved performance to separate solutes and/or dispersoidsfrom a liquid to be processed and possesses a membrane structureexcelling in biocompatibility. The asymmetric porous membrane thereforecan be suitably used particularly when the liquid to be processed isblood and is most suitably used as a separating membrane forextracorporeal circulation such as dialysis treatment.

BACKGROUND ART

Separation using a membrane has been commonly employed for separating orcondensing a specific solute from a multi-dispersion fluid consisting ofa solvent and many types of solutes and/or dispersoids (hereinaftersimply referred to as “solutes”). As the separation method, “sizebarrier separation” to separate a solute according to the size byproducing pores with a specific size in the membrane and “charge barrierseparation” to separate a solute using a charged membrane and causingthe solute to electrically repulse according to charges possessed by thesolute have been known. In addition, as the separation method using amembrane, methods utilizing the difference in properties exhibited bysolutes when treated with a membrane such as the adsorption force,ion-exchange capacity, and solubility-dispersiblity have been known.These separation methods are widely used in industries for desalting,water processing, food and pharmaceutical manufacturing, gas separation,and the like.

Blood purification therapy is carried out as a medical treatment forremoving various toxins accumulated in blood with an objective ofimproving diseases such as renal failure and hepatic failure. The methodof separation using a membrane have been applied to the Bloodpurification therapy. The blood purification therapy has a long historyin artificial kidneys used for treating chronic or acute renal failures.Various artificial kidney membranes using collodion flat membranes,hollow fiber membranes made from synthetic polymers, and the like havebeen put in to practice. A method utilizing a blood processing membranewith a larger pore diameter is also used for blood purification such asplasma exchange and fractionating blood plasma components.

These blood processing membranes for extracorporeal circulation includea dialysis membrane, filtration membrane, diafiltration membrane, andthe like. Suppressing pore blockage due to adsorption of plasma proteinsand preventing protein denaturation due to contact with the membrane aredemanded first of all for blood processing using a separating membrane.To this end, it has been necessary to make the membrane surfaceincluding pores coming in contact with blood hydrophilic.

On the other hand, efficiently removing wastes from blood is essentialfor a dialysis separating membrane of artificial kidneys used fortreating renal insufficiency diseases. In recent years, as a result ofthe process for identifying wastes to be removed and ascertainingsubstances causing various complications accompanying a long-term orshort-term dialysis, the substances to be removed now include, inaddition to low molecular weight compounds such as urea and ammonia thathave been removed in conventional dialysis, low molecular weight plasmaproteins such as β2-microglobulin (hereinafter referred to as β2MG) andadvanced glycation end products (hereinafter referred to as AGE).

In view of this situation, various high performance blood processingmembranes have been made commercially available. Major membranematerials include natural polymers such as regenerated cellulose and itsmodified product, cellulose polymers such as cellulose acetate, andsynthetic polymers such as a polyacrylonitrile-based polymer,polymethylmethacrylate-based polymer, polyamide-based polymer,polysulfone-based polymer, and ethylene-vinyl alcohol copolymer.

In terms of the structure, membranes are broadly classified intohomogeneous membranes with a dense structure as a whole andinhomogeneous (asymmetric) membranes consisting of a dense selectiveseparating layer and a porous supporting layer. From the viewpoint ofpermeability, the latter membranes are more preferable due to the leastpermeation resistance and the capability of ensuring physical membranestrength by the supporting layer.

Among these, a hydrophobic aromatic polysulfone-based polymer is gainingits position as a representative membrane material in recent years dueto the versatility as a resin material, strength as a structuralmaterial, resistance to sterilization treatment with heat or radiation,and superior controllability of the pore diameter and membrane structurewhen manufacturing the membrane. However, since the aromaticpolysulfone-based polymer is highly hydrophobic and, therefore, affectsthe blood clotting system, deaeration, and the like, this polymer isblended with hydrophilic polyvinyl pyrrolidone (hereinafter referred toas PVP) for use as a hollow separating membrane. This membrane has beenregarded to be free from the complement activity that has been reportedto occur when an untreated cellulose membrane comes into contact withblood and from the physiological activity harmful to the human body suchas anaphylaxis caused by bradykinin production that occurs underspecific conditions during dialysis using a polyacrylonitrile membranewith negative charges.

The PVP-blend polysulfone membrane can be manufactured in a wet spinningprocess comprising extruding a dope blend of an aromaticpolysulfone-based polymer and water soluble PVP from the outer cylinderof a cylindical nozzle, causing the spun material to come into contactwith an aqueous coagulant to effect phase separation, and removing thephase containing a large amount of PVP formed by the phase separationfrom the system.

Although it is possible to control the average pore size on the membranesurface that comes into contact with blood by changing the compositionof the aqueous coagulant in this method, the pore size distribution onthe surface of the resulting separating membrane tends to become widedue to fluctuations in the PVP molecular weight distribution and thepolymer concentration in the dope, the shear force when the dope isdischarged, and the like. For this reason, when low molecular weightplasma proteins are removed at a high ratio using this separatingmembrane, albumin which is a plasma protein useful for the human body isunnecessarily removed.

In addition, since a part of PVP that remains in the resultingseparating membrane must be removed using a large amount of solvent andtaking a long period of time in the membrane manufacturing process toprevent elution from the membrane during blood processing. This posesserious problems in the manufacturing process such as a decrease inproductivity and requirement for processing a large amount of wasteliquid.

In an effort to overcome these drawbacks, a method for separating plasmaproteins with different isoelectric points using a separating membranewith negatively charged groups introduced on the surface, simulating arenal glomerular basement membrane, has been investigated and developed.Okayama Medical Journal, Vol. 105, 317(1993) reports separation of threeplasma proteins with a molecular weight in the range of 14,300-66,000having different isoelectric points using a negatively charged membranefor dialysis made from an ethylene-vinyl alcohol copolymer with sulfonicacid groups introduced in the membrane surface. The report describesthat the sieving coefficient for plasma proteins with differentisoelectric points varies and permeation selectivity by negative chargescan be improved by increasing the quantity of negative charges in themembrane.

Japanese Patent Application Laid-open No. 5-131125 discloses that ahemodialysis membrane made from a blend of a sulfonated aromaticpolysulfone-based polymer and an aromatic polysulfone-based polymerexhibits a high sieving coefficient for β2MG and a low sievingcoefficient for albumin at the same time. In this manner, ultrafiltermembranes having negatively charged groups on the surface that comesinto contact with blood are known to exhibit high selective permeabilityfor plasma proteins.

However, as physiologically well known, negatively charged groups, whenbrought into contact with blood, activate the coagulation factor XII,one of the intrinsic clotting factors, and the resulting fragment XIIaactivates the coagulation factor XI in the presence of high molecularweight kininogen (e.g. E. Cenni, et al. “Biomaterials and BioengineeringHandbook” Chap. 8, 205, D. L. Wise ed., Mercel Dekker, New York, (2000)and Kidney Int. 1999 March 55(3) 11097-103). This activation acts as atrigger to activate the cascade for the intrinsic blood clotting system.The factor XIIa converts prekallikrein into kalliklein, which acts onhigh molecular weight kininogen to produce bradykinin (hereinafterabbreviated to BKN). The produced BKN induces an anaphylactoid reactionsuch as a slight fever and anesthesia of fingers and lips, when used inhemodialysis treatment. Therefore, direct contact of negatively chargedgroups with blood must be avoided in hemodialysis.

Japanese Patent Application Laid-open No. 8-505311 discloses a methodfor suppressing production of bradykinin using a membrane made from apolymer blend of a non-sulfonated polysulfone-based polymer and asulfonated aromatic polysulfone-based polymer, wherein the product ofthe sulfonation degree of the sulfonated polysulfone-based polymer andthe content of the sulfonated polysulfone-based polymer in the blend is100 or less. However, use of this method of reducing the total sulfonicacid residues results in a decrease in the selective permeability ofproteins, which is inevitably accompanied by a decrease in the cut offperformance. In addition, the degree of the bradykinin productioncontrol disclosed in the patent application is simply lower than in thecase where the polymer blend contains a large amount of sulfonatedpolysulfone. This does not necessarily indicate that the amount ofbradykinin production is decreased to the extent not affecting theliving body. Specifically, it is not known whether or not the amount ofbradykinin production is a level safe for use in artificial kidneys.

Japanese Patent Application Laid-open No. 11-313886 discloses a methodof using a neutral or cationic polymer for a semipermeable membrane fordialysis based on a negatively charged polyacrylonitrile to preventactivation of blood or plasma when coming in contact with thesemipermeable membrane. Because negative charges on the pore surfacesall over the membrane are covered with a neutral or cationic polymer inthis method, the amount of bradykinin production after the treatment isexpected to decrease. However, the membrane does not have sufficientfractionation performance due to small negative charges of thesemipermeable membrane for dialysis using the polyacrylonitrile as abase material.

In this manner, any conventional methods cannot provide a separatingmembrane having a sufficient cut off performance, while suppressing sideeffects on biological systems due to direct contact of the negativelycharged groups with blood.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide an asymmetric porousmembrane excelling in selective separation (cut off) performance ofplasma proteins, exhibiting almost no endogenous coagulation activity,complementary activity, and quinine activity, remarkably excelling inbiocompatibility, and usable in hemodialysis, plasma separation, and thelike. In particular, a first object of the present invention is toprovide an asymmetric porous membrane using, as a base material, asynthetic polymer that can separate human serum albumin with a molecularweight of about 67,000 from proteins with a molecular weight in therange of 30,000-40,000 represented by AGE at a high precision.

A second object of the present invention is to provide a method formanufacturing an asymmetric porous membrane having both biocompatibilityand selective separation capability.

In view of the above situation, the inventors of the present inventionhave conducted extensive studies on asymmetric porous membranes formedmainly from synthetic polymers. As a result, the inventors have foundthat the above objects can be achieved by providing a dense layersubstantially free from electric charges at least on the outermostsurface on the side on which a liquid to be processed is loaded and bycausing at least a part of the membrane other than the outermost surfaceto be negatively charged. This finding has led to the completion of thepresent invention.

In the present invention, the dense layer substantially free fromelectric charges present at least on the outermost surface functions asa size barrier (sieving function according to the molecular size) andthe part of the membrane other than the outermost surface functions as acharge barrier (sieving function according to repulsion of charges).

Therefore, the present invention relates to:

(1) an asymmetric porous membrane mainly formed from a synthetic polymerhaving a cross-sectional structure, wherein a dense layer substantiallyfree from electric charges is present at least on the outermost surfaceon the side on which a liquid to be processed is loaded and at leastpart of the membrane other than the outermost surface is negativelycharged,

(2) the asymmetric porous membrane described in (1) above, wherein thedense layer is substantially free from electric charges in its entirety,

(3) the asymmetric porous membrane described in (2) above, whereinnegative charges are densely present immediately below the dense layer,

(4) the asymmetric porous membrane described in (2) above, whereinnegative charges are present all over the membrane except for the denselayer,

(5) the asymmetric porous membrane described in any one of (1)-(4)above, wherein the negative charges are originated from a chargedpolymer different from the synthetic polymer forming the porousmembrane,

(6) the asymmetric porous membrane described in any one of (1)-(4)above, wherein the negative charges are originated from the syntheticpolymer mainly forming the part of the porous membrane excluding thedense layer,

(7) the asymmetric porous membrane described in (1) above, wherein onlythe outermost surface of the dense layer is substantially free fromelectric charges,

(8) the asymmetric porous membrane described in (7) above, wherein thenegative charges are densely present immediately below the outermostsurface layer,

(9) the asymmetric porous membrane described in (7) above, wherein thenegative charges are present all over the membrane except for theoutermost surface of dense layer,

(10) the asymmetric porous membrane described in any one of (7)-(9)above, wherein the negative charges are originated from a chargedpolymer different from the synthetic polymer forming the porousmembrane,

(11) the asymmetric porous membrane described in any one of (7)-(9)above, wherein the negative charges are originated from the syntheticpolymer mainly forming the part of the porous membrane excluding theoutermost surface of the dense layer,

(12) the asymmetric porous membrane described in (6) or (11) above,wherein the synthetic polymer possessing negative charges has a zetapotential at pH 7.4 of −2 mV or less, as measured for a substrate filmobtained from the polymer,

(13) the asymmetric porous membrane described in (12) above, wherein thesynthetic polymer possessing the negative charges is a polysulfone-basedpolymer containing at least one polymer selected from the groupconsisting of sulfonated polysulfone-based polymers and aliphaticpolysulfone-based polymers,

(14) the asymmetric porous membrane described in (13) above, wherein thesulfonated polysulfone-based polymers are at least one polymer selectedfrom the group consisting of sulfonated aromatic polysulfone-basedpolymers, sulfonated aliphatic polysulfone-based polymers, andsulfonated products of a copolymer of a hydrophilic polymer and anaromatic polysulfone-based polymer,

(15) the asymmetric porous membrane described in any one of (1)-(14)above, wherein the layer substantially free from electric charges ismade from a non-charged hydrophilic material,

(16) the asymmetric porous membrane described in (15) above, wherein thenon-charged hydrophilic material is at least one polymer selected fromthe group consisting of hydrophilic polymers, mixtures of a hydrophilicpolymer and an aromatic polysulfone-based polymer, and copolymers of ahydrophilic polymer and an aromatic polysulfone-based polymer,

(17) the asymmetric porous membrane described in (16) above, wherein thehydrophilic polymer is a linear or branched alkylene oxide polymer orpolyvinyl pyrrolidone,

(18) the asymmetric porous membrane described in any one of (1)-(17)above, wherein the membrane separates a plurality of solutes and/ordispersoids in the liquid to be processed by filtration and/ordiffusion,

(19) the asymmetric porous membrane described in (18) above, wherein themembrane is a membrane for dialyzing blood and/or a membrane forfiltering blood,

(20) a method for manufacturing the asymmetric porous membrane describedin any one of (1)-(5) above, comprising providing a porous substratemembrane with an asymmetric structure mainly made from a syntheticpolymer substantially free from electric charges and having a denselayer on the side on which a liquid is loaded and filtering or diffusinga solution of a negatively charged polymer that can be blocked by thedense layer from the side opposite to the dense layer to block thenegatively charged polymer from permeating through the dense layer,thereby introducing negative charges to the part excluding the denselayer and immobilizing the negatively charged material to the partexcluding the dense layer,

(21) the method described in (20) above, wherein the negative chargesare introduced at a high density immediately below the dense layer byblocking the negatively charged polymer immediately below the denselayer,

(22) a method for manufacturing the asymmetric porous membrane describedin anyone of (1), (2), (4), and (6)-(19) above, comprising forming aporous substrate membrane from a polymer solution containing a syntheticpolymer having negative charges as a main component, causing the surfaceof the substrate membrane to come in contact with a solution of asynthetic polymer substantially free from electric charges, andcoagulating the polymer to form a layer substantially free from electriccharges,

(23) a method for manufacturing the asymmetric porous membrane describedin any one of (1)-(19) above, comprising extruding a polymer solutioncontaining a synthetic polymer having negative charges as a maincomponent from the outer cylinder of a double cylindical spinneret andinjecting a solution of a synthetic polymer substantially free fromelectric charges and exhibiting an action of coagulating the abovesynthetic polymer from the inner cylinder of the double cylindicalspinneret, and

(24) a method for manufacturing the asymmetric porous membrane describedin any one of (1)-(19) above, comprising injecting a polymer solutioncontaining a synthetic polymer having negative charges as a maincomponent from the outer cylinder of a triple cylindical spinneret,injecting a solution of a synthetic polymer substantially free fromelectric charges from the middle cylinder of the triple cylindicalspinneret, and injecting a solvent exhibiting an action of coagulatingthe synthetic polymer having negative charges and the synthetic polymersubstantially free from electric charges from the inner cylinder of thetriple cylindical spinneret.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic sectional view showing one embodiment of theasymmetric porous membrane of the present invention.

FIG. 2 is a schematic view showing a method for introducing negativecharges to immediately below the dense layer of the asymmetric porousmembrane of the present invention.

FIG. 3 is a graph showing the sieving coefficient of α1-microglobulinand albumin for various membranes.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENT

Although any synthetic polymer commonly used as a separating membranecan be used without specific limitations for the synthetic polymermainly forming the asymmetric porous membrane of the present invention,synthetic polymers preferably used for processing blood is desirable.Specifically, the synthetic polymer is preferably selected frompolyacrylonitrile-based polymers, polymethylmethacrylate-based polymers,polyamide-based polymers, polysulfone-based polymers, and ethylene-vinylalcohol copolymers. Of these, polysulfone-based polymers are mostpreferable due to the excellent strength, superior resistance tosterilization treatment, and controllability in the pore diameter andstructure during membrane preparation.

The term “-based polymer” in the present invention refers to a syntheticpolymer containing that polymer as a main component. For example, apolyacrylonitrile-based polymer in the present invention refers to asynthetic polymer containing polyacrylonitrile as a main component.

In addition to the main component, the synthetic polymer may containmonomers having any optional functional group such as an anionic group,for example. In addition, a part of the polymer may be chemicallymodified to introduce a functional group such as an anionic group.

Since such a synthetic polymer is a main component for forming themembrane, it is possible to use other components in combination such asa hydrophilic polymer for providing hydrophilic properties or a poreforming agent.

In the present invention, a liquid loaded to the membrane is a liquidcontaining a plurality of solutes and/or dispersoids. A typical exampleis blood that contains various solutes with a low to high molecularweight as well as blood cells as dispersoids. Blood includes not onlywhole blood but also blood from which components such as plasma anderythrocytes have been separated. Therefore, typical examples of theasymmetric porous membrane of the present invention are membranes usablefor the purposes such as hemodialysis, hemofiltration, and plasmaseparation.

The asymmetric porous membrane mainly formed from a synthetic polymer ofthe present invention preferably has a cross-section structure having adense layer on the side on which a liquid to be processed is loaded anda supporting layer with a porous structure formed inside with a porediameter larger than the pore diameter of the dense layer, wherein theaverage pore diameter increases toward the side opposite to the side onwhich a liquid to be processed is loaded. The membrane may be either aflat membrane or a hollow fiber membrane with no specific limitations tothe configuration inasmuch as this structure can be maintained.

When the dense layer is thin, albumin that is a useful protein in bloodeasily permeates through the membrane, whereas when the dense layer isthick, the permeation resistance increases, giving rise to a decrease inthe total permeation amount. Therefore, the thickness of the dense layeris preferably about 1-20 μm, and more preferably 2-10 μm. The averagepore diameter of the dense layer should be determined to ensure anincrease in the permeability of low molecular weight plasma proteins andAGE that cause dialysis amyloidosis and a decrease in the amount ofleaking plasma albumin. Another important factor in determining the porediameter is to avoid contact of the supporting layer having negativecharges with the coagulation factor XII, high molecular weightkininogen, and prekallikrein. Therefore, the cut off molecular weight ofthe dense layer is preferably about 10-100 kD, and more preferably30-100 kD. The cut off molecular weight herein refers to the averagemolecular weight of dextran molecule of which the rate of blocking is90%.

The above dense layer must be substantially free from electric chargesat least on the outermost surface. The dense layer may be free fromelectric charges either in its entirety or only on the outermostsurface. The term “substantially free from electric charges” hereinrefers to the state of the layer having an electric charge of the ζpotential at pH 7.4 of −2 mV to 30 mV determined according to the ζpotential measurement of Examples. The term “outermost surface” hereindoes not refer to a dense layer or the like that can be macroscopicallyidentified by a cross-sectional photograph of membrane, but refers to athin layer that can be analyzed by a surface analysis means such asX-ray photoelectron spectroscopy.

The asymmetric porous membrane of the present invention must contain, inaddition to the dense layer functioning as a size barrier, negativecharges functioning as a charge barrier at least in a part of themembrane other than the outermost surface.

With regard to the negative charge distribution, when the dense layer isnot electrically charged in its entirety, the negative charges maybepresent in apart of or all over the membrane excluding the dense layer,but at least a part of the negative charges must function as a chargebarrier. Since the porous membrane of the present invention is anasymmetric porous membrane with a large average pore diameter on theside opposite to the side on which a liquid to be processed is loaded,if negative charges are present at least on the side on which theaverage pore diameter is close to that of the dense layer, that part isexpected to effectively function as a main charge barrier. If thenegative charges are present at a high density immediately below thedense layer, such negative charges are more preferable as a chargerbarrier. In the case of blood dialysis, negative charges are preferablypresent all over the membrane except for the dense layer to prevent areverse flow of physiological substances having negative charges such asendotoxin.

On the other hand, when only the outermost surface of the dense layerdoes not have negative charges, the negative charges may be presenteither in a part or all over the membrane excluding the dense layer, butat least a part of the negative charges must function as a chargebarrier. If negative charges are present at least in a part of themembrane other than the outermost surface of the dense layer, that partis expected to effectively function as a charge barrier. In the case ofhemodialysis, negative charges are present preferably all over themembrane except for the uppermost surface of the dense layer to preventa reverse flow of physiological substances having negative charges suchas endotoxin.

FIG. 1 shows an embodiment of the asymmetric porous membrane of thepresent invention. As indicated in FIG. 1, in the cross-section in theliquid permeation direction, a dense layer (a) with the smallest poresize is provided on the side coming in contact with the liquid to beprocessed. The dense layer preferably has a thickness of several mm orless to function as a resistance to permeation. A layer (b) withnegative charges is provided on the permeation side of the membraneimmediately below the dense layer. The pore radius of the charged layer(b) is larger than pore radius of the dense layer (a). Although thereare no specific limitations, the thickness of about 1 μm or more issufficient for the charged layer. In this embodiment, the dense layer(a) is substantially free from electric charges in its entirety.

The negative charges of which at least a part functions as a chargebarrier originate from a charged substance provided to a membrane afterformation when a formed membrane does not have electric charges or fromthe membrane itself when the membrane inherently has negative charges.

When the negative charges originate from a charged substance provided tothe membrane after formation, the membrane is prepared from anasymmetric porous membrane substantially free from electric charges as abase material by immobilizing a negatively charged polymer from the sideopposite to the dense layer of the base material membrane. When thenegative charges are provided to the base material membranesubstantially free from electric charges in this manner, at least a partof the negative charges functions as a charge barrier in a desired area.

When the negative charges originate from charges inherently possessed bythe base material membrane, it is sufficient that an outermost surfacesubstantially free from electric charges be formed on the surface ofthis membrane possessing the negative charges. As examples of such amembrane, a membrane prepared by coating a synthetic polymersubstantially free from electric charges on the surface of a dense layerof an asymmetric porous membrane possessing negative charges, acomposite membrane prepared by forming a dense layer of coagulatedsynthetic polymer substantially free from electric charges on thesurface of a porous membrane possessing negative charges, and the likecan be given. A membrane prepared by integrating a porous substrate filmpossessing negative charges and a dense layer of which at least theoutermost surface is substantially free from electric charges duringmembrane formation using a multiple slit-type spinneret may also beused. In addition, a membrane with negative charges provided on a partof the base material membrane can be obtained by changing thecompositions of the synthetic polymer extruded from two or more slits.These alternatives are appropriately selected. In this manner, when thebase material membrane is a porous membrane possessing negative charges,at least a part of the negative charges inherently possessed by themembrane functions as a charge barrier in a desired area of themembrane.

A substrate membrane possessing negative charges in the presentinvention may be a membrane formed mainly from any one of syntheticpolymers previously described (page 9, lines 4-11). Typically, such amembrane is an asymmetric porous membrane having an electric charge ofthe ζ potential at pH 7.4 of −2 mV or less, and preferably −4 mV or lessand −50 mV or more. Any appropriately prepared membranes or commerciallyavailable asymmetric porous membranes can be used irrespective of thetype of polymer or composition inasmuch as the above zeta potentialrequirement is satisfied.

The above-mentioned synthetic polymers possessing negative charges arethe polymers that can produce the substrate membrane exhibiting theabove zeta potential.

The substrate membrane substantially free from electric charges in thepresent invention may be formed mainly from any synthetic polymerdescribed above (page 14, lines 20-25) and refers to an asymmetricporous membrane having an electric charge of the ξ potential at pH 7.4of more than −2 mV, and preferably more than −2 mV and less than +30 mV.Any appropriately prepared membranes or commercially availableasymmetric porous membranes can be used irrespective of the type ofpolymer or composition inasmuch as the above zeta potential requirementis satisfied.

The above-mentioned synthetic polymers substantially free from electriccharges are the polymers that can produce the substrate membraneexhibiting the above zeta potential.

If the asymmetric porous membrane having such negative charges is used,the negative charges function as a charge barrier in the area other thanthe outermost surface having no charges to electrostatically repulsehuman serum albumin and suppress permeability of plasma albumin, wherebythe amount of plasma albumin leaked can be reduced.

The term “mainly formed from a synthetic polymer” herein indicates that50% or more of the components forming the asymmetric porous membrane isa specific polymer. For example, in the case of a polysulfone-basedpolymer, 50 wt % or more, and preferably 60% or more of the componentsforming the asymmetric porous membrane should be the polysulfone-basedpolymer.

The present invention will be hereinafter described on an embodiment inwhich the negative charges are originated from electric chargesinherently possessed by the substrate membrane and the synthetic polymeris a polysulfone-based polymer. However, the present invention shouldnot be limited to this embodiment.

The term “polysulfone-based polymer” in the present invention refers toall polymers having a sulfone bond and includes both sulfonated polymersand non-sulfonated polymers. Copolymers with a hydrophilic polymer arealso included. Here, the hydrophilic polymer includes a linear orbranched alkylene oxide-based polymer represented by polyethylene oxide,polyvinyl pyrrolidone, polyethylene glycol, and the like.

The polysulfone-based polymers are broadly classified into aromaticpolysulfone-based polymers and aliphatic polysulfone-based polymers. Theterm “aromatic polysulfone-based polymer” in the present inventionindicates non-sulfonated aromatic polysulfone-based polymersdistinguished from sulfonated aromatic polysulfone-based polymers. Inthe same manner, the term “aliphatic polysulfone-based polymer” in thepresent invention indicates non-sulfonated aliphatic polysulfone-basedpolymers distinguished from sulfonated aliphatic polysulfone-basedpolymers.

As specific examples of the aromatic polysulfone-based polymer used inthe present invention, aromatic polysulfone-based polymers containing arecurring unit of the following chemical formula (1), chemical formula(2), chemical formula (3), chemical formula (4), or chemical formula (5)can be given. Of these, the aromatic polysulfone-based polymerscontaining the recurring unit of the chemical formula (1), chemicalformula (2), or chemical formula (3), easily industrially available, arepreferable.

-   Chemical Formula (1):-   Chemical Formula (2):-   Chemical Formula (3):-   Chemical Formula (4):-   Chemical Formula (5):

Although there are no specific limitations to the degree ofpolymerization indicated by the symbol n in the above formulas, theweight average molecular weight of the polymer is preferably in therange of 1,000-1,000,000, and more preferably 5,000-100,000.

As specific examples of the aliphatic polysulfone-based polymer used inthe present invention, polymers containing the recurring unit shown bythe following chemical formula (6) can be given.

-   Chemical Formula (6):

Although there are no specific limitations to the degrees ofpolymerization indicated by the symbols m and 1 in the above chemicalformula (6), the weight average molecular weight of the polymer ispreferably in the range of 6,000-600,000, and more preferably10,000-200,000.

In the present invention, the polysulfone-based polymer may be used as acopolymer with a hydrophilic polymer. As example of the copolymer of anon-sulfonated polysulfone-based polymer and a hydrophilic polymer usedin the present invention, copolymers of a hydrophilic polymer and anaromatic polysulfone-based polymer, specifically block or graftcopolymers of a linear or branched polyalkylene oxide-based polymerrepresented by polyethylene oxide, polyvinyl pyrrolidone, polyethyleneglycol, or the like and the above aromatic polysulfone-based polymer canbe given. Of these, block or graft copolymers of a linear or branchedpolyalkylene oxide-based polymer represented by polyethylene oxide andthe above aromatic polysulfone-based polymer are preferable.

The copolymer of a non-sulfonated polysulfone-based polymer and ahydrophilic polymer used in the present invention includes, in additionto the above block or graft copolymers of a hydrophilic polymer and anaromatic polysulfone-based polymer, random copolymers of the recurringunits in the hydrophilic polymer and the recurring units in the aromaticpolysulfone-based polymer. Of these, the block or graft copolymers of ahydrophilic polymer and an aromatic polysulfone-based polymer are morepreferable.

In the present invention, the polysulfone-based polymer preferablycomprises at least one polymer selected from the sulfonatedpolysulfone-based polymers and aliphatic polysulfone-based polymers.

The sulfonated polysulfone-based polymer generally refers to asulfonated polymer having a sulfone bond and includes, but is notlimited to, sulfonated aromatic polysulfone-based polymers, sulfonatedaliphatic polysulfone-based polymers, and copolymers of these sulfonatedpolymers with a hydrophilic polymer.

As specific examples of these sulfonated aromatic polysulfone-basedpolymers and sulfonated aliphatic polysulfone-based polymers, sulfonatedcompounds of the above-mentioned specific polysulfone-based polymers aregiven and can be preferably used.

As examples of the copolymer with a hydrophilic polymer, copolymers of ahydrophilic polymer and a sulfonated product of aromaticpolysulfone-based polymer, specifically, block or graft copolymers of alinear or branched polyalkylene oxide-based polymer represented bypolyethylene oxide, polyvinyl pyrrolidone, polyethylene glycol, or thelike and a sulfonated product of the above aromatic polysulfone-basedpolymer can be given. Of these, block or graft copolymers of a linear orbranched polyalkylene oxide-based polymer represented by polyethyleneoxide and a sulfonated product of aromatic polysulfone-based polymer arepreferable.

The aromatic polysulfone-based polymer or aliphatic polysulfone-basedpolymer can be sulfonated using a known method. One example of such amethod, in the case of sulfonation of an aromatic polysulfone-basedpolymer, comprises reacting a solution of an aromatic polysulfone-basedpolymer in methylene chloride with a solution of chlorosulfonic acid inmethylene chloride while stirring in a reaction vessel to produce apolymer, precipitating the resulting polymer in isopropanol, and washingand drying the precipitate to obtain a polymer powder. However, theprocess for sulfonation is not limited to this method.

The method for synthesizing the copolymer of a hydrophilic polymer and asulfonated product of aromatic polysulfone-based polymer includes, butis not limited to, (a) a method of sulfonating a copolymer of anaromatic polysulfone-based polymer and a hydrophilic polymer, (b) amethod of sulfonating an aromatic polysulfone-based polymer andcopolymerizing the resulting sulfonated polymer with a hydrophilicpolymer, and (c) a method of sulfonating raw material monomers for anaromatic polysulfone-based polymer, synthesizing a sulfonated aromaticpolysulfone-based polymer, and copolymerizing the sulfonated aromaticpolysulfone-based polymer with a hydrophilic polymer.

When the sulfonated polysulfone-based polymer thus obtained bysulfonation is a sulfonated aromatic polysulfone-based polymer with areplacement degree of one or more, the hydrophilic properties due tosulfonation are so strong that the resulting polymer tends to becomewater-soluble and is difficult to use.

When the replacement degree is from 0.5 or more to less than 1.0, thesulfonated aromatic polysulfone-based polymer is swellable with waterand cannot be used alone. Such a polymer must be used mixed with anaromatic polysulfone-based polymer which is a non-sulfonatedpolysulfone-based polymer. In this case, the ratio by weight of thesulfonated aromatic polysulfone-based polymer to the aromaticpolysulfone-based polymer in the dope solution is preferably 0.02-0.75,and more preferably 0.05-0.5.

When the replacement degree is from 0.05 or more to less than 0.5, thesulfonated aromatic polysulfone-based polymer may be used either aloneor mixed with an aromatic polysulfone-based polymer. When used as amixture, the ratio by weight of the sulfonated aromaticpolysulfone-based polymer to the aromatic polysulfone-based polymer inthe dope is preferably 0.1-1, and more preferably 0.1-0.9. It ispossible to have a desired amount of negative charges (sulfonationdensity) in the whole membrane by changing the replacement degree andthe mixing ratio. The static repulsion due to negative charges can thusbe adjusted. If the replacement degree is less than 0.05, the amount ofnegative charges by sulfonation is too low to cause the membrane toexhibit sufficient cutt off performance even if the sulfonated aromaticpolysulfone-based polymer is used alone.

On the other hand, when the replacement degree is less than 0.3, sincehydrophilicity is insufficient the case where the sulfonated aromaticpolysulfone-based polymer is used alone or mixed with an aromaticpolysulfone-based polymer, a hydrophilic polymer must be used incombination to increase the hydrophilicity. Here, as the hydrophilicpolymer to be used in combination, a linear or branched alkyleneoxide-based polymer represented by polyethylene oxide, polyvinylpyrrolidone, polyethylene oxide, or the like, and a copolymer of thehydrophilic polymer and an aromatic polysulfone-based polymer can begiven.

Using a hydrophilic polymer in combination to increase hydrophilicityirrespective of the replacement degree is a preferable embodiment of theasymmetric porous membrane of the present invention. The amount of thehydrophilic polymer in the dope solution is preferably 0.5-20 wt %, andmore preferably 1-10 wt %. The replacement degree (degree of sulfonationor DS) herein refers to the number of sulfonic acid groups present perrecurring unit of the polysulfone skeleton.

Among the polysulfone-based polymers, aliphatic polysulfone-basedpolymers have strong negative charges without being sulfonated. Forexample, the aliphatic polysulfone-based polymer comprising therecurring unit of the chemical formula (6) may be sulfonated by theabove-described known method, but can be used in place of the sulfonatedpolysulfone-based polymer without being sulfonated.

The sulfonated aliphatic polysulfone-based polymers and the aliphaticpolysulfone-based polymers may be used either individually or incombination with an aromatic polysulfone-based polymer. When used incombination, the ratio by weight of the sulfonated aliphaticpolysulfone-based polymer or the aliphatic polysulfone-based polymer tothe aromatic polysulfone-based polymer in the dope solution ispreferably 0.1 to 0.9, and more preferably 0.15 to 0.8, depending on thenegative charges possessed by the sulfonated aliphatic polysulfone-basedpolymer and the aliphatic polysulfone-based polymer.

Using a hydrophilic polymer in combination to increase hydrophilicity isa preferable embodiment. The amount of the hydrophilic polymer in thedope solution is preferably 0.5-20 wt %, and more preferably 1-10 wt %.

The sulfonated aromatic polysulfone-based polymers, sulfonated aliphaticpolysulfone-based polymers, and aliphatic polysulfone-based polymers canbe used in combination as components to form the asymmetric porousmembrane possessing negative charges of the present invention.

A dense layer free from electric charges is present at least on theoutermost surface on the liquid loading side of the asymmetric porousmembrane of the present invention. That outermost surface preferablycomprises a non-charged hydrophilic material. Specifically, when thedense layer is free from electric charges in its entirety, either thedense layer may be formed from the non-charged hydrophilic material orthe outermost surface substantially free from electric charges maycomprise the non-charged hydrophilic material. When only the outermostsurface of the dense layer does not have electric charges, thisoutermost surface is preferably formed from the non-charged hydrophilicmaterial.

In the present invention, the non-charged hydrophilic material refers toa hydrophilic material substantially free from electric charges. When amaterial is substantially free from electric charges, that material hasan electric charge of the ζ potential at pH 7.4 of more than −2 mV andless than +30 mV determined according to the ζ potential measurementdescribed in Examples.

Specifically, even if a material possesses negative charges, such amaterial is included in the non-charged hydrophilic material of thepresent invention, inasmuch as an anaphylactoid reaction such as aslight fever and anesthesia of fingers and lips, induced by bradykinin(BKN) produced by contact with blood, does not occur during ahemodialysis treatment, specifically, inasmuch as the degree of negativecharges in terms of the ζ potential at pH 7.4 determined according tothe ζ potential measurement is more than −2 mV.

The non-charged hydrophilic material will now be explained in moredetail taking the case where a polysulfone-based polymer is used as thesynthetic polymer as an example. However, the present invention is notlimited to this example.

The hydrophilic material refers to a synthetic or naturally occurringpolymer or a derivative thereof exhibiting affinity with water moleculevia a hydrogen bond-type functional group such as a hydroxyl group,acrylamide group, or ether group or an electrolytically dissociatingfunctional group such as a carboxyl group, sulfonic acid group, orquaternary amino group. Examples include naturally occurring polymersand oligomers such as starch, pectin, gelatin, casein, and dextran;semisynthetic polymers and oligomers such as methyl cellulose,carboxymethyl cellulose, and hydroxyethyl cellulose; linear or branchedpolyalkylene oxides such as polyethylene oxide; polyethylene glycol,polyvinyl alcohol, polyvinyl methyl ether, polyvinyl pyrrolidone, sodiumpolyacrylate, polyethyleneimine, and polyacrylamide; a mixture of atleast one of these polymers and oligomers and an aromaticpolysulfone-based polymer; and a copolymer of at least one of thesepolymers and oligomers and an aromatic polysulfone-based polymer.

Of these, hydrophilic polymers such as linear or branched polyalkyleneoxides, polyethylene glycol, and polyvinyl pyrrolidone, a mixture of thehydrophilic polymer and aromatic polysulfone-based polymer, and acopolymer of the hydrophilic polymer and aromatic polysulfone-basedpolymer are preferable, with more preferable hydrophilic materials beinglinear or branched alkylene oxide-based polymers, a mixture of thealkylene oxide-based polymer and aromatic polysulfone-based polymer, acopolymer of the alkylene oxide-based polymer and aromaticpolysulfone-based polymer, polyvinyl pyrrolidone, a mixture of polyvinylpyrrolidone and aromatic polysulfone-based polymer, and a copolymer ofpolyvinyl pyrrolidone and aromatic polysulfone-based polymer.Particularly preferable hydrophilic materials are polyethylene oxide,polyvinyl pyrrolidone, mixtures of these polymers and aromaticpolysulfone-based polymer, and copolymers of these polymers and aromaticpolysulfone-based polymer.

In the present invention, the polyalkylene oxide used as a non-chargedhydrophilic material or a component for the copolymer with thepolysulfone-based polymer is not limited to a linear polyalkylene oxide,but a branched polyalkylene oxide may also be used. The polyalkyleneoxides can provide the substrate membrane with superior biocompatibilitydue to their capability of forming a diffusive layer on the membranesurface and can significantly suppress contact of proteins such as highmolecular weight kininogen with the substrate membrane. The method formanufacturing aromatic polysulfone copolymers having the branchedpolyalkylene oxide is disclosed in U.S. Pat. No. 6,172,180, for example.

The linear or branched alkylene oxide-based polymer, mixture of thealkylene oxide-based polymer and aromatic polysulfone-based polymer, thecopolymer of the alkylene oxide-based polymer and aromaticpolysulfone-based polymer, polyvinyl pyrrolidone, mixture of polyvinylpyrrolidone and aromatic polysulfone-based polymer, and copolymer ofpolyvinyl pyrrolidone and aromatic polysulfone-based polymer,particularly polyethylene oxide, polyvinyl pyrrolidone, mixtures of thepolyethylene oxide or polyvinyl pyrrolidone and aromaticpolysulfone-based polymer, and copolymers of the polyethylene oxide orpolyvinyl pyrrolidone and aromatic polysulfone-based polymer can exhibitextremely low activation of platelet, endogenous coagulation system,complementary system, and kuinine system when brought into contact withblood, possess excellent biocompatibility, and permit adhesion of only avery small amount of plasma proteins. These materials can thus suppresschanges in permeability over time as a blood processing membrane duringdialysis, for example.

These non-charged hydrophilic materials are dissolved at a mixing ratioin a wide range not only in good solvents for polysulfone-based polymersforming the porous membrane as the substrate membrane, but also in mixedsolvents consisting of the good solvent and nonsolvents for thepolysulfone-based polymers. Therefore, it is possible to form a surfacelayer of these non-charged hydrophilic materials on the blood contactside of the substrate membrane by dissolving them in the mixed solventwhen preparing the substrate membrane or by applying the solution to thesubstrate membrane after preparation.

The presence or absence of a layer substantially free from electriccharges on the outermost surface of the dense layer can be evaluated inthe present invention by appropriately selecting a surface analysismethod such as X-ray photoelectron spectroscopy according to the type ofsynthetic polymer forming the substrate membrane and the type of chargedpolymer. For example, when the synthetic polymer is a polysulfone-basedpolymer, the presence or absence of a layer containing a non-chargedhydrophilic material as the layer substantially free from electriccharges on the outermost surface of the dense layer can be evaluated asfollows.

A ratio of oxygen atoms to sulfur atoms ([O]/[S]) or a ratio of nitrogenatoms to sulfur atoms ([N]/[S]) is determined by X-ray photoelectronspectroscopy (hereinafter referred to as “XPS”) as a surfaceconcentration index of the non-charged hydrophilic material. Here,either [O]/[S] or [N]/[S] is selected according to the type of thenon-charged hydrophilic material. When the polymer contains polyvinylpyrrolidone, for example, a value of [N]/[S] is used. When the polymercontains polyalkylene oxide, a value of [O]/[S] is used. When thepolymer contains both polyvinyl pyrrolidone and polyalkylene oxide,either one of the values [N]/[S] and [O]/[S] is used.

Possessing a layer containing a non-charged hydrophilic material in thepresent invention indicates that, when the surface concentration indexof the non-charged hydrophilic material is [O]/[S], an inequality[O]/[S]>6, or preferably [O]/[S]>7 is satisfied; and when the surfaceconcentration index of the non-charged hydrophilic material is [N]/[S],an inequality [N]/[S]>1.5, or preferably [N]/[S]>2.0 is satisfied. Whenthe polymer contains both polyvinyl pyrrolidone and polyalkylene oxide,it is sufficient that either the inequality for the value [N]/[S] or[O]/[S] is satisfied.

The method for manufacturing the asymmetric porous membrane will now bedescribed. There are broadly seven methods for manufacturing theasymmetric porous membrane of the present invention.

Specifically, they are:

(1) a method of using a porous membrane having negative charges as asubstrate membrane and forming a dense layer by coagulating a syntheticpolymer substantially free from electric charges on the surface of thesubstrate membrane,

(2) a method of providing a solution containing a charged polymer with asize impermeable through the dense layer from the supporting layer sidetoward the dense layer side of the substrate membrane of an asymmetricporous membrane substantially free from electric charges, therebyimmobilizing the charged polymer substance in the membrane.

(3) a method of extruding a dope solution containing a synthetic polymerhaving negative charges from an outer cylinder of a double cylindicalspinneret, extruding a solution of a synthetic polymer substantiallyfree from electric charges in a mixed solvent containing a nonsolventand a good solvent from the inner cylinder of the double cylindicalspinneret, and coagulating the synthetic polymer to form a membrane,

(4) a method of injecting the dope solution from an outer cylinder atriple cylindical spinneret, injecting a solution containing a syntheticpolymer substantially free from electric charges from a middle cylinderof the triple cylinder spinneret nozzle, and injecting a solventexhibiting an action of coagulating the synthetic polymer substantiallyfree from electric charges from an inner cylinder of the triple cylinderspinneret nozzle to form a membrane,

(5) a method of extruding the dope solution from the outer cylinder of adouble cylindical spinneret, extrudung a mixture of a nonsolvent andgood solvent from the inner cylinder, coagulating the synthetic polymerto form a porous membrane, and causing the dense layer contact surfaceof the resulting hollow membrane to come in contact with a solutioncontaining a synthetic polymer substantially free from electric charges,thereby forming a membrane,

(6) a method of using an asymmetric porous membrane substantially freefrom electric charges as a substrate membrane, separately forming adense layer by coagulating a synthetic polymer having negative chargeson the surface of the substrate membrane, and causing a solution of asynthetic polymer substantially free from electric charges to come incontact with the surface of the dense layer, and

(7) a method of extruding the dope solution from a middle cylinder of atriple cylindical spinneret, extruding a solution containing a syntheticpolymer substantially free from electric charges from an outer cylinderof the triple cylindical spinneret, and extruding a solvent exhibitingan action of coagulating the synthetic polymer substantially free fromelectric charges from an inner cylinder of the triple cylindicalspinneret to form a dense layer containing negative charges, and causinga solution of a synthetic polymer substantially free from electriccharges to come in contact with the surface of the dense layer, therebyforming a membrane.

Among the above methods, the methods (1) and (2) are preferably appliedto the case where the dense layer does not have electric charges allover that layer. The methods (3) to (7) are preferably applied to thecase where the outermost surface of the dense layer does not haveelectric charges.

The method (1) comprises providing a porous membrane having negativecharges but not a desired dense layer and separately forming a denselayer by coagulating a synthetic polymer substantially free fromelectric charges on the surface of the porous membrane.

The substrate membrane may be formed mainly from any synthetic polymerdescribed above (page 9, lines 4-17). Typically, such a membrane is aporous membrane having a ζ potential at pH 7.4 of −2 mV or less, andpreferably −4 mV or less and −50 mV or more. Any appropriately preparedmembranes or commercially available porous membranes can be usedirrespective of the type of polymer or composition inasmuch as the abovezeta potential requirement is satisfied.

A known composite membrane manufacturing method can be applied toformation of the dense layer. Specifically, a solution of the syntheticpolymer forming the dense layer in a good solvent for the syntheticpolymer but not dissolving the substrate membrane is used. After causingthe solution to come in contact with the surface of the substratemembrane, that surface is then caused to come in contact with acoagulating fluid that is a nonsolvent for the synthetic polymer formingthe dense layer and does not dissolve the substrate membrane, or thesolvent is removed by drying to cause the polymer to deposit, whereby acomposite membrane can be obtained.

The dense layer provided on the surface of the substrate membrane may beformed from any one of the synthetic polymers previously described (page9, lines 4-17). A synthetic polymer substantially free from electriccharges is used. Any type of polymer and composition can be used withoutspecific limitations inasmuch as the above definition can be satisfied.In addition, a non-charged hydrophilic material may be applied to theoutermost surface of the formed dense layer or the dense layer itselfmay be formed from the non-charged hydrophilic material.

To form a charged layer immediately below the dense layer in theasymmetric porous membrane in the method (2), a solution of a chargedmaterial is supplied by filtration or diffusion from the supportinglayer side of the membrane to the dense layer side. If a chargedsubstance with a size that cannot permeate through the dense layer isused, the charged substance permeating through the supporting layer ofthe membrane toward the dense layer is blocked by the dense layer andcaptured by the porous supporting layer immediately below the denselayer. The captured charged substance is then immobilized.

To immobilize the charged substance, a solution containing acrosslinking agent (immobilizing agent) that can cause a crosslinkingreaction among molecules of the charged substance or between themolecules of the charged substance and molecules of the membranematerial is supplied by permeation or diffusion from the supportinglayer side of the membrane to cause local crosslinking reactions,thereby physically or chemically immobilizing the charged substanceimmediately below the dense layer of the membrane. Although thecrosslinking is a preferable method, any other method that canimmobilize the charged substance in the membrane can be used.

Alternatively, the crosslinking agent (immobilizing agent) may besupplied by diffusion from the dense layer side, while supplying thecharged substance by filtration or diffusion from the supporting layerside of the membrane, to immobilize the charged substance immediatelybelow the dense layer.

In the step of coagulating the asymmetric membrane, it is possible toreact the coagulating agent that is present outside the membranedirectly with the polymer solution using a chemical compound that canintroduce charged functional groups. Distribution of charges in thecross-section direction of the membrane can be polarized so that chargesmay be present outside the membrane, but do not occur in the denselayer. A charged membrane in which charges do not expose inside can beobtained in this manner.

Although the substrate membrane in this method may be formed from anyone of the synthetic polymers previously described (page 9, lines 4-11),this membrane is an asymmetric porous membrane substantially free fromelectric charges. A membrane substantially free from electric chargesrefers to the membrane having an electric charge of ζ potential at pH7.4 of more than −2 mV and less than +30 mV determined according to theζ potential measurement described in the Examples. Any appropriatelyprepared membranes or commercially available asymmetric porous membranescan be used irrespective of the type of polymer or composition inasmuchas the above zeta potential requirement is satisfied.

Although the charged polymer having negative charges provided to thesubstrate membrane may be any naturally occurring or synthetic chargedpolymers, polymers having a sulfonic acid groups are particularlypreferable due to a large degree of electrolytic dissociation underphysiological pHs. Specifically, any sulfated polysaccharidesrepresented by acidic mucopolysaccharides such as heparin, heparansulfate, chondroitin sulfate, and kerato sulfate, and semi-syntheticpolysaccharides such as dextran sulfate can be used. The syntheticpolymer that can be used includes copolymers produced from vinyl-typemonomers containing sulfonic acid groups such as sodiummethallylsulfonate. Water-soluble polymers are preferable for thepurpose of not damaging the substrate membrane during processing. Thesepolymers are particularly preferable due to their chemical structurethat enables the polymers to crosslink by themselves by processing witha crosslinking reagent or by irradiation or to be immobilized byreacting with part of membrane.

In addition, the charged polymers with a molecular size impermeablethrough a dense layer are used and caused to be present in the membraneso that the negative charges do not expose on the surface of the denselayer. Furthermore, if a charged polymer having high affinity with thesubstrate membrane, particularly having high adsorptivity, is used, amembrane with negative charges provided all over the substrate membraneexcept for the dense layer can be obtained.

It is possible to apply a non-charged hydrophilic material to theoutermost surface of the dense layer also in the manufacturing method(2).

The manufacturing methods (3)-(5) will now be described taking the casesin which a polysulfone-based polymer is used as the synthetic polymerand a non-charged hydrophilic material is used as the synthetic polymersubstantially free from electric charges as examples. Of course, thepresent invention is not limited to these examples.

As the polymer forming the dope solution used for the manufacture of theasymmetric porous membrane, which is the substrate membrane, (1) asulfonated polysulfone-based polymer alone, (2) a mixture of asulfonated polysulfone-based polymer and an aromatic polysulfone-basedpolymer, (3) an aliphatic polysulfone-based polymer alone, (4) a mixtureof a aliphatic polysulfone-based polymer and aromatic polysulfone-basedpolymer, (5) a mixture of the polymer or polymer composition of eitherone of (1)-(4) and a hydrophilic polymer, and the like can be given. Oneof these polymers or polymer compositions is appropriately selectedtaking the membrane performance into consideration. The dope solution isprepared by dissolving these polymers or polymer compositions in asolvent. The solvent dissolving the polysulfone-based polymers ishereinafter referred to as a good solvent.

As the good solvent, N,N-dimethylacetamide, N,N-dimethylformamide,N-methyl-2-pyrrolidone, dimethylsulfoxide, and the like are preferablyused, with N,N-dimethylformamide and N-methyl-2-pyrrolidone beingparticularly preferable. These good solvents are not required to be usedalone, but two or more of them may be used in combination to adjust thesolubility of the polymer or the viscosity of the dope solution or tocontrol the membrane performance. In addition, it is possible to add anonsolvent for the polysulfone-based polymers, such as water, alcoholssuch as isopropyl alcohol and ethanol, inorganic salts such as sodiumchloride and calcium chloride, and glycols such as propylene glycol,tetraethylene glycol, and polyethylene glycol (hereinafter referred toas “nonsolvent”) to accelerate pore formation that affects the membraneperformance or to prevent void formation. The type and amount of thenonsolvents are appropriately selected and adjusted according to therequired performance of the porous membrane.

Although the concentration of the polymer in the dope solution dependson the molecular weight of the polymer, that concentration is in therange of 10-50 wt %, and preferably 15-40 wt % from the viewpoint ofdrawability and membrane strength.

In the manufacturing method (3), the dope solution from the outercylinder is used as a spinning solution. A mixture of the solvent forthe dope solution used for the outer cylinder and a nonsolvent is usedfor the solution containing the non-charged hydrophilic materialextruded from the inner cylinder. As the nonsolvent, water, isopropylalcohol, ethanol, propylpropylene glycol, tetraethylene glycol, and thelike can be given. Of these, water is preferable. The mixing ratio ofthe good solvent and nonsolvent is the largest factor to determine theaverage pore diameter of the substrate membrane. In the case of theporous membrane made mainly from a polysulfone-based polymer, anincrease in the ratio of water that is a nonsolvent generally tends todecrease the average pore size of the dense layer. Therefore, the ratioof the good solvent to the nonsolvent is preferably from 10/90 to 65/35,and more preferably from 20/80 to 55/45.

In the porous membrane formed by coagulating polymer solutionssimultaneously extruded from the outer cylinder and inner cylinder as inthe manufacturing method (3), the surface layer formed close to theoutermost surface of the dense layer of the porous membrane may peel offor elimination during post-processing and the like. However, when thepolymer solutions are simultaneously extruded from the outer cylinderand the inner cylinder, polymers entangle among molecular chains in theinterface of the outer cylinder solution and the inner cylinder solutionwhile coagulating, whereby such peeling or elimination is prevented.Even if a mixed solution, in which a hydrophilic polymer such aspolyalkylene oxide or polyvinyl pyrrolidone is used alone as thenon-charged hydrophilic material, is used as the inner cylindersolution, peeling or elimination does not occur if the hydrophilicpolymer has a weight average molecular weight of 5,000 or more, andpreferably 8,000 or more. On the other hand, when the average molecularweight of the hydrophilic polymer used as the non-charged hydrophilicmaterial is less than 5,000, a mixture of the hydrophilic polymer and anaromatic polysulfone-based polymer or a copolymer of the hydrophilicpolymer and an aromatic polysulfone-based polymer is preferably used tothe extent that such a polymer mixture or copolymer is dissolved in themixed solvent used for the inner cylinder.

The concentration of the non-charged hydrophilic material that is animportant factor for determining the thickness of the surface layer isabout 0.01-15 wt %, and preferably 0.05-5 wt %. This range ofconcentration ensures not only the thickness of the surface layersufficiently large to suppress influence of negative charges on thesubstrate membrane, but also formation of a thin layer with a uniformthickness. In addition, the above concentration range ensures a lowviscosity of the solution that can accelerate difussion of polymer tothe outer cylinder solution side, enabling the polymer solution touniformly cover the pore surface without changing the pore size near thesurface of the substrate membrane.

The hollow fiber membrane can be manufactured using a triple cylindricalspinneret as in the manufacturing method (4). In the manufacturingmethod (4), an asymmetric porous membrane is manufactured by extrudingthe dope solution from an outer cylinder of the triple cylindicalspinneret, a solution containing a non-charged hydrophilic material froma middle cylinder, and a solvent exhibiting an action of coagulating thepolysulfone-based polymer and the non-charged hydrophilic material froman inner cylinder.

As the solvent used for the solution of the non-charged hydrophilicmaterial extruded from the middle cylinder, N,N-dimethylacetamide,N,N-dimethylformamide, N-methyl-2-pyrrolidone, dimethylsulfoxide, andthe like are preferably used, with N,N-dimethylformamide andN-methyl-2-pyrrolidone being particularly preferable. These solvents arenot required to be used alone, but two or more of them may be used incombination to adjust the solubility of the polymer in the solution orthe viscosity of the solution or to control the membrane performance. Inaddition, it is possible to add a nonsolvent including water, alcoholssuch as isopropyl alcohol and ethanol, inorganic salts such as sodiumchloride and calcium chloride, and glycols such as propylene glycol,tetraethylene glycol, and polyethylene glycol to accelerate the poreformation that affects the membrane performance or to prevent the voidformation. The type and amount of the nonsolvents are appropriatelyselected and adjusted according to the required performance of theporous membrane.

A mixture of a good solvent and a nonsolvent is extruded from the innercylinder. As the good solvent, N,N-dimethylacetamide,N,N-dimethylformamide, N-methyl-2-pyrrolidone, dimethylsulfoxide, andthe like are preferably used, with N,N-dimethylformamide andN-methyl-2-pyrrolidone being particularly preferable. These solvents arenot required to be used alone, but two or more of them may be used incombination. As the nonsolvent, water, isopropyl alcohol, ethanol,propylpropylene glycol, tetraethylene glycol, and the like can be given.Of these, water is most preferable. The mixing ratio of the good solventand nonsolvent is the largest factor to determine the average porediameter of the substrate membrane. In the case of the porous membranemade mainly from a polysulfone-based polymer, an increase in the ratioof water that is a nonsolvent generally tends to decrease the averagepore size of the dense layer. Therefore, the ratio of the solvent to thenonsolvent is preferably from 10/90 to 65/35, and more preferably from20/80 to 55/45.

Hollow membranes spun by the method (3) or (4) are coagulated in acoagulating bath, washed, and dried to obtain the asymmetric porousmembrane of the present invention. A post-processing using hightemperature vapor or the like is effective to increase hydrophilicity ofthe outermost surface of the dense layer. Water that is a nonsolvent ispreferably used for the coagulating bath. The water may contain asolvent such as N,N-dimethylacetamide, N,N-dimethylformamide,N-methyl-2-pyrrolidone, or polyvinylpyrrolidone. These solvents are notrequired to be used alone, but two or more of them may be used incombination to adjust polymer coagulating performance or to controlmembrane performance. A nonsolvent such as an alcohol, for example,isopropyl alcohol or ethanol, may be added. The type and amount of thenonsolvents are appropriately selected and adjusted according to therequired performance of the porous membrane. The temperature of thecoagulating bath is important since the coagulating bath temperaturesignificantly affects the membrane performance. The temperature ispreferably in the range of 20-90° C., and more preferably 50-70° C.

The substrate membrane used in the method (5) can be prepared using amethod almost the same as in the methods (3) and (4). In this case, thesame solvent composition of a good solvent and a nonsolvent as describedin the manufacturing method (4) as the mixed solvent used for the innercylinder of the triple cylindical spinneret can be applied to thesolvent used as the inner cylinder solvent during spinning. A porousmembrane as a substrate membrane is formed. After drying, the denselayer surface of the porous membrane is caused to come in contact with asolution containing a non-charged hydrophilic material to form thesurface layer.

The solution containing a non-charged hydrophilic material has a ratioof the solvent to the nonsolvent preferably from 5/95 to 65/35, and morepreferably from 20/80 to 55/45. The concentration of the non-chargedhydrophilic material is 0.1-15 wt %, and preferably 0.05-5 wt %. Thereare no specific limitations to the method for causing the dense layersurface of the porous membrane to come in contact with the solutioncontaining a non-charged hydrophilic material. In a typical method,after preparing a hollow fiber module by a known method, a solutioncontaining the non-charged hydrophilic material is fed to the denselayer surface side of the module, then the solution is replaced withwater until dissolution does not occur any more, thereby coagulating andimmobilizing the non-charged hydrophilic material, and the coagulatednon-charged hydrophilic material is dried, as required. In this case,the surface is preferably maintained to be in contact with the solutionfor a period of time long enough to cause the surface of the substratemembrane to become swelled or the temperature is maintained higher thanroom temperature, preferably at about 50-70° C., to prevent peeling orelimination of the surface layer.

EXAMPLES

The present invention will be described in more detail by examples,which should not be construed as limiting the present invention.

Evaluation Methods

(Measurement of Replacement Degree)

The mol number of sulfonic acid groups is determined by theneutralization titration method described in the Analytical ChemistryManual (1971 second edition, edited by The Japan Society for AnalyticalChemistry, p 367, 2-47-3 Quantitative Analysis). The weight percentageof polysulfone skeleton is determined from the integral values of thearomatic part and the methylene chain part of polyethylene oxide in NMR.The value is converted into a value per recurring unit of polysulfoneskeleton. The number of sulfonic acid groups, that is the replacementdegree (degree of sulfonation or DS), is then determined from theresulting value and the previously determined amount of sulfonic acidgroups.

(Method of Measuring Weight Average Molecular Weight)

The molecular weight was measured by a measuring instrument (System-21,manufactured by Shodex Co.) linked to GPC columns (KD-806M, KD-803,KD-802 manufactured by Showdex Co.) using dimethylacetamide (DMAc) as adeveloping solution at a column temperature of 50° C. at a flow rate of1 ml/min. A converted molecular weight was calculated using apolystyrene standard sample (TSK standard polystyrene, manufactured byTosoh Corp.).

(Method of Measuring Cut Off Molecular Weight)

The internal diameter of a hollow fiber membrane was measured. Thenumber of hollow fibers to obtain constant performance was counted usingthe following formula. A module with an effective length of 18 cm wasprepared by bonding both ends using epoxy adhesive. Before the test, thehollow fibers were thoroughly washed with a physiological salinesolution for injection (Otsuka Normal Saline, manufactured by OtsukaPharmaceutical Co., Ltd.).Number of threads=Flow rate/(π/4)×(internal diameter/10,000)²×linearvelocity×60 min)wherein the linear velocity is 1 cm/sec and the flow rate is 2.0 ml/min.

Next, 10 g of dextran 40,000 (manufactured by Sigma Corp., Mw=41,272)and 10 g of dextran 70,000 (manufactured by Sigma Corp., Mw=71,000) weredissolved in a physiological saline solution for injection (OtsukaNormal Saline, manufactured by Otsuka Pharmaceutical Co., Ltd.). Thedextran solutions were heated to 37° C. and caused to pass through themodule at a flow rate of 2.0 ml/min. When the dextran solution flowedout of the outlet of the module, a pressure was applied to maketransmembrane pressure (TMP) 25 mmHg. After 10 minutes, the filtrate wascollected for five minutes and was used as the sample for evaluation.Ultrafiltration rate (UFR, ml/mmHg·m²·hr) was calculated from the amountof the obtained filtrate. The evaluation sample and the dextran solutionused for performance test were analyzed by HPLC under the followingconditions. Column: (Analytical column) Shodex GF-710HQ, (Guard column)GF-1G-7B, column temperature: 40° C., detector: RI (Shimadzu RID-6A),mobile phase: physiological saline solution, flow rate: 0.3 ml/min, sixstandard dextrans (Mw=186,000, 100,000, 48,000, 23,700, 12,200, and5,800) were used to prepare a molecular weight calibration curve andconvert the holding time of chromatogram into the dextran molecularweight. A peak strength in the chromatogram for each evaluation samplewas divided by the peak strength in the chromatogram for dextransolution to determine a sieve coefficient (SC). The block rate isdefined as (1-SC)×100.

(Method of Measuring ζ Potential of Porous Membrane)

The ζ potential was measured using an electrokinetic analyzer (EKA)manufactured by Anton Paar GmbH as follows. The EKA pump was set to avoltage of 20V. A measuring sample was placed in the center ofcylindrical cell in a width of 3-5 cm by sandwiching between Ag/AgClelectrodes so that a pressure of −930 to −950 mba is applied. 500 ml ofa 0.001 mol/l KCl aqueous solution at 25° C. was prepared from a 0.01mol/l KCl solution for testing (manufactured by Kishida Chemical Co.)and distilled water (manufactured by Otsuka Pharmaceutical Co., Ltd.).After filling the entire measuring circuit with the KCl solution, a 0.1mol/l KOH solution (for volumetric analysis, manufactured by Wako PureChemical Industries, Ltd.) was added to adjust the pH to 11. Then, the ζpotential of the hollow fiber membrane was measured each time the pHchanges by 0.8 in the range of pH 11-3, while titrating the 0.1 mol/lHCl solution (for volumetric analysis, manufactured by Wako PureChemical Industries, Ltd.) using RTU (Remote Controlled Titration Unit(manufactured by Anton Paar GmbH)).

(Method of Measuring XPS)

Hollow fiber membranes were cut and opened to expose the innerside.Several hollow fiber membranes cut in this manner were aligned within ameasurable eyesight to measure XPS using an XPS apparatus (PHI-5400manufactured by Physical Electronics Inc.) under the followingconditions.

Excitation source: MgKα (15 kV/26.7 mA), analysis area: 3.5 mm×1 mm,intake region: Survey Scan (for qualitative analysis) 1,100-0 eV, NarrowScan (for quantitative analysis and chemical analysis) Cls, Ols, S2p,Pass Energy: Survey Scan: 178.9 eV and Narrow Scan: 35.75 eV. Theelement concentration was calculated from the area strength of theobtained Narrow Scan spectrum using a library relative sensitivitycoefficient of the apparatus. The XPS was calculated using the resultingelement concentration. The relative sensitivity coefficient used wasCls: 0.296, Ols: 0.711, S2p: 0.666, and N1s: 0.477.

(Test Method for Lactate Dehydrogenase (LDH) and Protein AdsorptionAmount as Platelet Activation Indexes)

A minimodule made from 56 hollow fibers with an effective length of 15cm (membrane area: 50 mm²), both ends being bonded using an epoxyadhesive was washed by feeding 10 ml of a physiological saline solution(Otsuka Normal Saline, manufactured by Otsuka Pharmaceutical Co., Ltd.)both inside and outside of the hollow fibers (hereinafter referred to as“priming”). Heparinized human blood placed in a 7 ml syringe pump wascharged to the module at a flow rate of 1.2 ml/min, followed by washingwith 10 ml of the physiological saline solution for each of inside andoutside the follow fibers. Hollow fibers were collected from the washedmodule, 28 for LDH determination and 23 for adsorbed protein, each witha length of 14 cm, and finely cut to be used as samples for measurement.

0.5 ml of Triton X-100/PBS solution (0.5 vol), obtained by dissolvingTriton X-100 (manufactured by Nakalai Tesque, Inc.) in a phosphatebuffer solution (PBS) (manufactured by Wako Pure Chemical Industries,Ltd.), was added to a spitz tube for measuring LDH. After a ultrasonictreatment for 60 minutes, 0.1 ml of the extract was reacted with 3 ml ofan LDH reaction reagent (LDH monotest, manufactured by BoehringerMannheim). Immediately after the reaction, 0.5 ml of the reactant wassampled to measure absorbance at 340 nm. The remaining reactant wasreacted for a further one hour at 37° C. and the absorbance at 340 nmwas measured to determine the decrease in the absorbance. The absorbancefor the unreacted membrane was measured in the same manner to determineΔ340 nm=(Absorbance immediately after sample reaction—Absorbance 60minutes after sample reaction)—(Absorbance immediately after blankreaction—Absorbance 60 minutes after blank reaction). The larger therate of decrease, the higher the LDH activity of the membrane.

2 ml of 1 vol % SDS/PBS solution obtained by dissolving sodium laurylsulfate (SDS) (manufactured by Nakalai Tesque Inc.) in PBS was added toa bottle for measuring adsorbed protein and the mixture was stirred atroom temperature for four hours. The extract was filtered through a 0.45μm filter paper. 0.2 ml of the filtrate was used as the test solution. 3ml of a bicinchoninic acid (BCA) protein assay reagent (manufactured byPierce Biotechnology, Inc.) was added to the test solution. The mixturewas reacted at 37° C. for 30 minutes to measure absorbance at 562 nm.Absorbance was measured in the same manner for the membrane not reactedwith blood, to deduct the resulting value from the absorbance of thetest solution. A calibration curve was prepared from the straight lineof standard protein absorbance to determine the amount of proteinabsorbance of the test solution.

(Measuring Method for Bradykinin (BKN))

100 sample fibers, each with a length of 16 cm, were bonded with siliconadhesive and a polyvinylchloride cover was attached to the outerperiphery (membrane area: 100 m²). After priming with purified water anda physiological saline solution in that order, the sample was heated ina hot water bath at 37° C. Heparinized human fresh heparin-added bloodwas fed using a syringe pump to cause the blood to come in contact withthe inner layer of the fibers. 5 ml of the blood at the outlet port wasrecovered in a test tube (containing 2 ml of an inhibitor manufacturedby SRL; components: trasylol, a soybean trypsin inhibitor, protaminesulfate, and EDTA-2Na) as a sample. The blood flow rate was 0.44 ml/minto ensure a contact time of four minutes. The blood was then cooled andcentrifuged to collect plasma, which was lyophilized for storing.Radioimmunoassay (RIA method) was employed for the measurement.

(Evaluation Method for Fractionation Performance Using Calf Serum)

Sieving coefficients of albumin (Mw=66,000), α1 microglobulin (α1MG andMw=33,000), and β2 microglobulin (β2MG and Mw=11,800) as indexes forfractionation performance were determined by the following methods.

The internal diameter of a membrane was measured. The number of fibersto obtain a membrane area of 120 mm² was counted. Both ends of thefibers were bonded with an epoxy adhesive to make an effective length of15 cm. The fibers were sufficiently washed with a physiological salinesolution to prepare a mini-module for the test. Calf serum (alyophilized product, manufactured by Valley Biomedical and Inc.) heatedand melted at 37° C. was diluted with a physiological saline solution tomake a total protein concentration 6.5 g/dense layer. α1 microglobulin(8 mg/l) (α1-M high grade Eiken, manufactured by Eiken Chemical Co.,Ltd.) and β2 microglobulin (5 mg/l) (β2-M high grade Eiken, manufacturedEiken Chemical Co., Ltd.) were added to make a serum test sample. Theserum was heated at 37° C. and filtered through the module at a flowrate of 1 ml/min. A pressure was applied to make TMP=34 mm Hg. After 60minutes, the filtrate was collected for use as the sample forevaluation. UFR (ml/mmHg·m²·hr) was calculated from the amount of theobtained filtrate. An albumin coloring reagent was added and absorbanceat 630 nm was measured to identify albumin filtration. The albumin SCwas calculated by dividing the absorbance of raw serum by the absorbanceof sample. A full automatic immunochemical analyzer (LX-6000,manufactured by Eiken Chemical Co., Ltd.) was used for determination ofSC of α1 microglobulin and β2 microglobulin.

Reference Example 1

(Preparation of Sulfonated Aromatic Polysulfone-Based Polymer)

Sodium bis(2-chlorobenzenesulfonate)-5,5′-sulfonate was synthesizedaccording to the method described in J. Polym. Sci., Part A: Polym.Chem., 31, 853-858 (1993).

A 1,000 ml three-necked separable flask was charged with 29.02 g ofbisphenol A (Tokyo Kasei Kogyo Co., Ltd.), 31.56 g of4,4′-dichlorodiphenylsulfone (Tokyo Kasei Kogyo Co., Ltd.), 10.23 g ofthe above sodium bis(2-chlorobenzenesulfonate)-5,5′-sulfonate, 52.76 gof potassium carbonate (Wako Pure Chemical Industries, Ltd.), 80.8 ml oftoluene (Wako Pure Chemical Industries, Ltd.), and 194.6 ml ofN-methyl-2-pyrrolidone (Tokyo Kasei Kogyo Co., Ltd.). The atmosphere inthe flask was replaced with nitrogen while stirring the mixture for twohours. After maintaining the mixture at 155° C., toluene was refluxedfor three hours, while removing water produced by azeotropicdistillation from the mixture using a deanstack trap. The mixture washeated to 190° C. After removing toluene, the mixture was maintained at190° C. for five hours. The reaction mixture was cooled to roomtemperature and 10,000 ml of distilled water was slowly added dropwisewhile stirring to obtain a fibrous branched polysulfone-based polymer.The residue obtained by filtration was poured into 5,000 ml of distilledwater. Concentrated hydrochloric acid was added to make the mixture pH2, followed by filtration. The filtrate was washed until pH reaches 7.After washing with 6,000 ml of 40% ethanol aqueous solution at 70° C.for three hours, the mixture was filtered. The residue was washed withethanol and dried at 50° C. under vacuum to obtain a sulfonated aromaticpolysulfone-based polymer with a degree of sulfonation (DS) of 0.3. Theyield was 65 g. The weight average molecular weight of the resultingpolymer was 75,000.

Reference Example 2

(Preparation of Block Copolymer of Branched Polyethylene Oxide andAromatic Polysulfone-Based Polymer)

A 1,000 ml three-necked separable flask was charged with 29.02 g ofbisphenol A (Tokyo Kasei Kogyo Co., Ltd.), 43.08 g of4,4′-dichlorodiphenylsulfone (Tokyo Kasei Kogyo Co., Ltd.), 50.00 g ofpotassium carbonate (Wako Pure Chemical Industries, Ltd.), 50 ml oftoluene (Wako Pure Chemical Industries, Ltd.), and 130 ml ofN-methyl-2-pyrrolidone (Tokyo Kasei Kogyo Co., Ltd.). The atmosphere inthe flask was replaced with nitrogen while stirring the mixture. Aftermaintaining the reaction mixture at 155° C., toluene was refluxed forthree hours, while removing water produced by azeotropic distillationfrom the reaction mixture using a deanstack trap. The reaction mixturewas heated to 190° C. After removing toluene, the mixture was maintainedat 190° C. for four hours to obtain a polysulfone prepolymer withchlorine atoms bonded at both ends. A 1,000 ml three-necked separableflask was charged with 129.86 g of polyethylene glycol #4000 (TokyoKasei Kogyo Co., Ltd., hydroxyl value 36 mg KOH/g), 26.72 g oftetra-functional block copolymer obtained by progressive addition ofpropylene oxide and ethylene oxide to ethylenediamine (BASF, Tetronic304: hydroxyl value 68 mg KOH/g), 200.00 g of potassium carbonate (WakoPure Chemical Industries, Ltd.), 150 ml of toluene (Wako Pure ChemicalIndustries, Ltd.), and 350 ml of N-methyl-2-pyrrolidone (Tokyo KaseiKogyo Co., Ltd.). The atmosphere in the flask was replaced with nitrogenwhile stirring the mixture. After maintaining the reaction mixture at155° C., toluene (Wako Pure Chemical Industries, Ltd.) was refluxed forthree hours, while removing water produced by azeotropic distillationfrom the reaction mixture using a deanstack trap. The reaction mixturewas heated to 190° C. After removing toluene by evaporation, 4.91 g of4,4′-difluorodiphenylsulfone (Tokyo Kasei Kogyo Co., Ltd.) was added.The mixture was maintained at 190° C. for six hours to obtain a branchedpolyethylene oxide prepolymer. The branched polyethylene oxideprepolymer was added to the reaction mixture of the above polysulfoneprepolymer with chlorine atoms bonded to both ends. The mixture wasmaintained at 190° C. for eight hours in a nitrogen atmosphere. Thereaction mixture was slowly added dropwise to 10,000 ml of distilledwater while stirring to obtain fibrous branched PEO-polysulfonecopolymer. The residue obtained by filtration was poured into 5,000 mlof distilled water. Concentrated hydrochloric acid was added to make themixture pH 2, followed by filtration. The filtrate was washed until pHreaches 7. After washing with 6,000 ml of a 40% ethanol aqueous solutionat 70° C. for three hours, the mixture was filtered. The residue waswashed with ethanol and dried at 50° C. under vacuum to obtain a blockcopolymer of branched polyethylene oxide and an aromatic polysulfone.The yield was 151.66 g. The ζ potential of the resulting copolymer was−0.3 mV and the weight average molecular weight was 60,000.

Reference Example 3

(Preparation of Graft Sulfonated Polysulfone)

200 g of aromatic polysulfone (UDEL P-1700 manufactured by AmocoEngineering Polymers Inc.) was irradiated with γ-rays at 1 Mrd/hr for 10hours in a dry ice atmosphere and added to a solution of 30 g of3-sulfopropyl methacrylate (a grafting agent) in 1 kg of a 3:1 mixtureof H₂O and t-BOH. The mixture was reacted at 40° C. for four hours in anitrogen atmosphere. After the reaction, the reaction product was washedwith alcohol, then with water, and dried to obtain a grafted sulfonatedpolysulfone for preparation of a dope. The degree of sulfonation of theresulting polymer was 0.2.

Reference Example 4

(Preparation of Ethylene Sulfone-Propylene Sulfone Copolymer)

4.9 g of ethylene sulfide and 14.0 g of propylene sulfide (bothmanufactured by Kanto Kasei Co., Ltd.) were mixed with 254 mg of ethylacetate in which 44.8 mg of magnesium perchlorate (manufactured by WakoPure Chemical Industries, Ltd.) was dissolved. The mixture was stirredfor five hours at 70° C. in a sealed container. The reaction product wasdissolved in 40 ml of 1-methyl-2-pyrrolidone (manufactured by Wako PureChemical Industries, Ltd.) and added to 1,000 ml of ethanol to obtain awhite precipitate of a polymer.

The precipitate was sufficiently washed with ethanol and ethanol wasremoved under reduced pressure at 60° C. to obtain 14.0 g ofpolysulfide. 1 g of the polysulfide was dissolved in 60 ml of1-methyl-2-pyrrolidone. A mixture of 4 ml of a 30% hydrogen peroxidesolution and 20 ml of formic acid was slowly added dropwise whilestirring. The mixture immediately generated exothermic heat and thesulfide was oxidized into sulfone to produce a precipitate of anethylene sulfone-propylene sulfone copolymer. The precipitate waspurified by repeating a washing procedure consisting of centrifugationfollowed by replacing the supernatant three times and dried at 60° C.under reduced pressure for four hours to obtain 0.85 g of a white solidof ethylene sulfone-propylene sulfone copolymer (aliphatic polysulfoneof the chemical formula 6). The weight average molecular weight of theresulting copolymer was 78,000.

Reference Example 5

(Preparation of Branched Polyethylene Oxide-Sulfonated PolysulfoneCopolymer)

Sodium bis(2-chlorobenzenesulfonate)-5,5′-sulfonate was synthesizedaccording to the method described in J. Polym. Sci., Part A: Polym.Chem., 31, 853-858 (1993).

A 1,000 ml three-necked separable flask was charged with 29.02 g ofbisphenol A (Tokyo Kasei Kogyo Co., Ltd.), 36.61 g of4,4′-dichlorodiphenylsulfone (Tokyo Kasei Kogyo Co., Ltd.), 11.86 g ofthe above sodium bis(2-chlorobenzenesulfonate)-5,5′-sulfonate, 52.76 gof potassium carbonate (Wako Pure Chemical Industries, Ltd.), 80.8 ml oftoluene (Wako Pure Chemical Industries, Ltd.), and 194.6 ml ofN-methyl-2-pyrrolidone (Tokyo Kasei Kogyo Co., Ltd.). The atmosphere inthe flask was replaced with nitrogen while stirring the mixture for twohours. After maintaining the mixture at 155° C., toluene was refluxedfor three hours, while removing water produced by azeotropicdistillation from the mixture using a deanstack trap. The mixture washeated to 190° C. After removing toluene, the mixture was maintained at190° C. for five hours to obtain both end-chlorinated sulfonatedpolysulfone (hereinafter referred to as polysulfone) prepolymer.

A 1,000 ml three-necked separable flask was charged with 129.84 g ofpolyethylene glycol #4000 (Tokyo Kasei Kogyo Co., Ltd., hydroxyl value36 mg KOH/g), 26.74 g of tetra-functional block copolymer obtained byprogressive addition of propylene oxide and ethylene oxide toethylenediamine (BASF, Tetronic 304: hydroxyl value 68 mg KOH/g), 200.03g of potassium carbonate, 173.0 ml of toluene, and 340.6 ml ofN-methyl-2-pyrrolidone. The atmosphere in the flask was replaced withnitrogen while stirring the mixture for two hours. After maintaining themixture at 155° C., toluene was refluxed for three hours, while removingwater produced by azeotropic distillation from the mixture using adeanstack trap. Next, 9.83 g of 4,4′-difluorodiphenylsulfone (TokyoKasei Kogyo Co., Ltd.) dissolved in 50 g of N-methyl-2-pyrrolidone wasadded. The mixture was heated to 190° C. After removing toluene over onehour, the mixture was maintained at 190° C. for five hours to obtain abranched polyethylene oxide prepolymer.

The reaction mixture of the branched polyethylene oxide prepolymer wasadded to the reaction mixture of the both end-chlorinated sulfonatedprepolymer, followed by the addition of 100 ml of toluene. Theatmosphere was replaced with nitrogen. After maintaining the reactionmixture at 155° C., toluene was refluxed for three hours, while removingwater produced by azeotropic distillation from the mixture using adeanstack trap. The mixture was heated to 190° C. After removing tolueneover one hour, the mixture was maintained at 190° C. for eight hours toobtain a branched polyethylene oxide-sulfonated polysulfone copolymer.The reaction mixture was slowly added dropwise to 10,000 ml of distilledwater while stirring to obtain a fibrous branched polyethyleneoxide-sulfonated polysulfone copolymer. The residue obtained byfiltration was poured into 5,000 ml of distilled water. Concentratedhydrochloric acid was added to make the mixture pH 2, followed byfiltration. The filtrate was washed until pH reaches 7. After stirringfor washing in 6,000 ml of a 40% ethanol aqueous solution at 70° C. forthree hours, the mixture was filtered. The residue was washed withethanol and dried at 50° C. under vacuum to obtain a branchedpolyethylene oxide-sulfonated polysulfone copolymer. The yield was 215g. The degree of sulfonation (DS) of the resulting polymer was 0.3 andweight average molecular weight was 37,000.

Examples for manufacturing asymmetric porous membranes using a doublespinneret nozzle or triple spinneret nozzle are shown in Examples 1-7.

Example 1

A spinning solution consisting of 18 parts by weight of an aromaticpolysulfone (UDEL P-1700 manufactured by Amoco Engineering PolymersInc.), 7 parts by weight of a sulfonated polysulfone copolymer preparedaccording to the method described in Reference Example 1, 15 parts byweight of tetraethylene glycol, and 60 parts by weight ofN-methyl-2-pyrrolidone was prepared. This spinning solution wasmaintained at 50° C. and extruded from a double cylindrical spinneretsimultaneously with an inner cylinder fluid consisting of 50 parts byweight of water, 49.5 parts by weight of N-methyl-2-pyrrolidone, and 0.5part by weight of a block copolymer of branched polyethylene oxide andan aromatic polysulfone prepared according to the method described inReference Example 2. The extruded hollow fiber was caused to run througha hood saturated with water vapor at an average temperature of 40° C.,immersed in water at 55° C. in a spinning bath installed 600 mm belowthe spinneret, and wound around a bobbin at a rate of 50 m/min. Thewound hollow fiber was washed with hot water at 90° C. for 90 minutesand dried at 70° C. to obtain hollow fiber for evaluation. The hollowfiber obtained was evaluated according to the above-described method.The results are shown in Table 1.

Example 2

A spinning solution consisting of 15 parts by weight of an aromaticpolysulfone (UDEL P-1700 manufactured by Amoco Engineering PolymersInc.), 7 parts by weight of a sulfonated polysulfone copolymer preparedaccording to the method described in Reference Example 1, 8 parts byweight of a block copolymer of branched polyethylene oxide and anaromatic polysulfone prepared according to the method described inReference Example 2, 10 parts by weight of tetraethylene glycol, and 60parts by weight of N-methyl-2-pyrrolidone was prepared. This spinningsolution was maintained at 50° C. and extruded from a double cylindricalspinneret simultaneously with an inner cylinder fluid consisting of 50parts by weight of water, 49.5 parts by weight ofN-methyl-2-pyrrolidone, and 0.5 part by weight of a block copolymer ofbranched polyethylene oxide and an aromatic polysulfone preparedaccording to the method described in Reference Example 2. The extrudedhollow fiber was caused to run through a hood saturated with water vaporat an average temperature of 40° C., immersed in water at 55° C. in aspinning bath installed 600 mm below the spinneret, and wound around abobbin at a rate of 50 m/min. The wound hollow fiber was washed with hotwater at 90° C. for 90 minutes and dried at 70° C. to obtain a hollowfiber for evaluation. The hollow fiber obtained was evaluated accordingto the above-described method. The results are shown in Table 1.

Example 3

A spinning solution consisting of 18 parts by weight of a graftsulfonated polysulfone prepared according to the method described inReference Example 3, 30 parts by weight of tetraethylene glycol, and 52parts by weight of N-methyl-2-pyrrolidone was prepared. This spinningraw fluid was maintained at 50° C. and extruded from a doublecylindrical spinneret simultaneously with an inner cylinder fluidconsisting of 50 parts by weight of water, 49.5 parts by weight ofN-methyl-2-pyrrolidone, and 0.5 part by weight of a block copolymer ofbranched polyethylene oxide and an aromatic polysulfone preparedaccording to the method described in Reference Example 2. The extrudedhollow fiber was caused to run through a hood saturated with water vaporat an average temperature of 40° C., immersed in water at 55° C. in abath installed 600 mm below the spinneret, and wound around a bobbin ata rate of 50 m/min. The wound hollow fiber was washed with hot water at90° C. for 90 minutes and dried at 70° C. to obtain a hollow fiber forevaluation. The hollow fiber obtained was evaluated according to theabove-described method. The results are shown in Table 1.

Example 4

A spinning solution consisting of 5 parts by weight of an ethylenesulfone-propylene sulfone copolymer (an aliphatic polysulfone of thechemical formula 6) prepared according to the method described inReference Example 4, 18 parts by weight of an aromatic polysulfone (UDELP-1700 manufactured by Amoco Engineering Polymers Inc.), 15 parts byweight of tetraethylene glycol, and 62 parts by weight ofN-methyl-2-pyrrolidone was prepared. The aliphatic polysulfone was usedfor this spinning solution as is without sulfonating, since thepolysulfone had a strong negative charge. This spinning solution wasmaintained at 50° C. and extruded from a double cylindrical spinneretsimultaneously with an inner cylinder fluid consisting of 50 parts byweight of water, 49.5 parts by weight of N-methyl-2-pyrrolidone, and 0.5part by weight of a block copolymer of branched polyethylene oxide andan aromatic polysulfone prepared according to the method described inReference Example 2. The extruded hollow fiber was caused to run througha hood saturated with water vapor at an average temperature of 40° C.,immersed in water at 55° C. in a bath installed 600 mm below thespinneret, and wound around a bobbin at a rate of 50 m/min. The woundhollow fiber was washed with hot water at 90° C. for 90 minutes anddried at 70° C. to obtain a hollow fiber for evaluation. The hollowfiber obtained was evaluated according to the above-described method.The results are shown in Table 1.

Example 5

A spinning solution consisting of 18 parts by weight of an aromaticpolysulfone (UDEL P-1700, manufactured by Amoco Engineering PolymersInc.), 7 parts by weight of a sulfonated polysulfone prepared accordingto the method described in Reference Example 1, 15 parts by weight oftetraethylene glycol, and 60 parts by weight of N-methyl-2-pyrrolidonewas prepared. This spinning solution was maintained at 50° C. andextruded from a double cylindrical spinneret nozzle simultaneously withan inner cylinder fluid consisting of 50 parts by weight of water and 50parts by weight of N-methyl-2-pyrrolidone. The extruded hollow fiber wascaused to run through a hood saturated with water vapor at an averagetemperature of 40° C., immersed in water at 55° C. in a spinning bathinstalled 600 mm below the spinneret, and wound around a bobbin at arate of 50 m/min. The wound hollow fiber was washed with hot water at90° C. for 90 minutes and dried at 70° C. to obtain a hollow fiber. Amini-module was prepared from the hollow fiber. After priming bythoroughly charging a mixed solution of 70 parts by weight of water at50° C., 29.5 parts by weight of N-methyl-2-pyrrolidone, 0.5 part of ablock copolymer of branched polyethylene oxide and an aromaticpolysulfone prepared according to the method described in ReferenceExample 2 into the hollow fiber, the mini-module was washed with waterand dried to obtain the hollow fiber for evaluation. The hollow fiberobtained was evaluated according to the above-described method. Theresults are shown in Table 1.

Example 6

A spinning solution for extruding from an outer cylinder of a triplespinneret nozzle consisting of 18 parts by weight of aromaticpolysulfone (UDEL P-1700 manufactured by Amoco Engineering PolymersInc.), 7 parts by weight of a sulfonated polysulfone prepared accordingto the method described in Reference Example 1, 10 parts by weight oftetraethylene glycol, and 65 parts by weight of N-methyl-2-pyrrolidonewas prepared and maintained at 50° C. Another spinning solution forextruding from a middle cylinder of the triple cylindrical spinneretconsisting of 18 parts by weight of an aromatic polysulfone (P-1700manufactured by Amoco Performance Products Inc.), 7 parts by weight of ablock copolymer of branched polyethylene oxide and an aromaticpolysulfone prepared according to the method described in ReferenceExample 2, 10 parts by weight of tetraethylene glycol, and 65 parts byweight of N-methyl-2-pyrrolidone was prepared and maintained at 50° C.These spinning solution were extruded from the triple cylindricalspinneret simultaneously with an inner cylinder fluid consisting of 50parts by weight of water and 50 parts by weight ofN-methyl-2-pyrrolidone. The extruded hollow fiber was caused to runthrough a hood saturated with water vapor at an average temperature of40° C., immersed in water at 60° C. in a bath installed 1,100 mm belowthe spinneret, and wound around a bobbin at a rate of 50 m/min. Thewound hollow fiber was washed with hot water at 90° C. for 90 minutesand dried at 70° C. to obtain a hollow fiber for evaluation. The hollowfiber obtained was evaluated according to the above-described method.The results are shown in Table 1.

Example 7

A spinning solution consisting of 5 parts by weight of a branchedpolyethylene oxide-sulfonated polysulfone copolymer prepared accordingto the method described in Reference Example 5, 18 parts by weight of anaromatic polysulfone (UDEL P-1700 manufactured by Amoco EngineeringPolymers Inc.), 15 parts by weight of tetraethylene glycol, and 62 partsby weight of N-methyl-2-pyrrolidone was prepared. This spinning solutionwas maintained at 50° C. and extruded from a double spinneretcylindrical simultaneously with an inner cylinder fluid consisting of 50parts by weight of water, 49.5 parts by weight ofN-methyl-2-pyrrolidone, and 0.5 part by weight of a block copolymer ofbranched polyalkylene oxide and an aromatic polysulfone preparedaccording to the method described in Reference Example 2. The extrudedhollow fiber was caused to run through a hood saturated with water vaporat an average temperature of 40° C., immersed in water at 55° C. in abath installed 600 mm below the spinneret, and wound around a bobbin ata rate of 50 m/min. The wound hollow fiber was washed with hot water at90° C. for 90 minutes and dried at 70° C. to obtain a hollow fiber forevaluation. The hollow fiber obtained was evaluated according to theabove-described method. The results are shown in Table 1.

Comparative Example 1

A homogeneous spinning solution consisting of 18 parts by weight of anaromatic polysulfone (UDEL P-1700 manufactured by Amoco EngineeringPolymers Inc.), 7 parts by weight of polyvinyl pyrrolidone, and 76 partsby weight of N-methyl-2-pyrrolidone was prepared. This spinning solutionwas maintained at 50° C. and extruded from a double cylindricalspinneret simultaneously with an inner cylinder fluid consisting of 50parts by weight of water and 50 parts by weight ofN-methyl-2-pyrrolidone. The extruded hollow fiber was caused to runthrough a hood saturated with water vapor at an average temperature of40° C., immersed in water at 55° C. in a bath installed 600 mm below thespinneret, and wound around a bobbin at a rate of 50 m/min. The woundhollow fiber was washed with hot water at 90° C. for 90 minutes, dippedin a 20 wt % glycerol aqueous solution at 60° C. for one hour, and driedat 70° C. to obtain a hollow fiber for evaluation. The hollow fiberobtained was evaluated according to the above-described method. Theresults are shown in Table 1.

Comparative Example 2

The same spinning solution as in Example 1 was maintained at 50° C. andextruded from a double cylindrical spinneret simultaneously with aninner cylinder fluid consisting of 50 parts by weight of water and 50parts by weight of N-methyl-2-pyrrolidone. The extruded hollow fiber wascaused to run through a hood saturated with water vapor at an averagetemperature of 40° C., immersed in water at 55° C. in a spinning bathinstalled 600 mm below the spinneret, and wound around a bobbin at arate of 50 m/min. The wound hollow fiber was washed with hot water at90° C. for 90 minutes and dried at 70° C. to obtain a hollow fiber forevaluation. The hollow fiber obtained was evaluated according to theabove-described method. The results are shown in Table 1.

Comparative Example 3

The same spinning solution as in Example 2 was maintained at 50° C. andextruded from a double cylindrical spinneret simultaneously with aninner cylinder fluid consisting of 50 parts by weight of water and 50parts by weight of N-methyl-2-pyrrolidone. The extruded hollow fiber wascaused to run through a hood saturated with water vapor at an averagetemperature of 40° C., immersed in water at 55° C. in a bath installed600 mm below the spinneret, and wound around a bobbin at a rate of 50m/min. The wound hollow fiber was washed with hot water at 90° C. for 90minutes and dried at 70° C. to obtain a hollow fiber for evaluation. Thehollow fiber obtained was evaluated according to the above-describedmethod. The results are shown in Table 1.

Comparative Example 4

The same spinning solution as in Example 3 was maintained at 50° C. andextruded from a double cylindrical spinneret simultaneously with aninner cylinder fluid consisting of 50 parts by weight of water and 50parts by weight of N-methyl-2-pyrrolidone. The extruded hollow fiber wascaused to run through a hood saturated with water vapor at an averagetemperature of 40° C., immersed in water at 55° C. in a bath installed600 mm below the spinneret, and wound around a bobbin at a rate of 50m/min. The wound hollow fiber was washed with hot water at 90° C. for 90minutes and dried at 70° C. to obtain a hollow fiber for evaluation. Thehollow fiber obtained was evaluated according to the above-describedmethod. The results are shown in Table 1.

Comparative Example 5

The same spinning solution as in Example 4 was maintained at 50° C. andextruded from a double spinneret nozzle simultaneously with an innercylinder fluid consisting of 50 parts by weight of water and 50 parts byweight of N-methyl-2-pyrrolidone. The extruded hollow fiber was causedto run through a hood saturated with water vapor at an averagetemperature of 40° C., immersed in water at 55° C. in a bath installed600 mm below the spinneret, and wound around a bobbin at a rate of 50m/min. The wound hollow fiber was washed with hot water at 90° C. for 90minutes and dried at 70° C. to obtain a hollow fiber for evaluation. Thehollow fiber obtained was evaluated according to the above-describedmethod. The results are shown in Table 1.

TABLE 1 Amount of adsorbed Cutt off LDH protein BKN β2MG α1MG Albumin MWζ Potential*³ (IU/m²) (mg/m²) production*¹ SC SC SC (Mw × 10⁻³) XPS*²(mV) Example 1 1.2 1.4 2.1 1.00 0.22 0.004 40 ± 5 7.8 −4 Example 2 1.01.3 1.9 1.00 0.24 0.004 40 ± 5 8.5 −3 Example 3 1.2 1.3 2.1 1.00 0.180.005 40 ± 5 8.0 −3 Example 4 2.0 1.8 2.3 1.00 0.18 0.005 40 ± 5 7.1 −5Example 5 1.3 1.5 2.2 1.00 0.22 0.004 40 ± 5 7.9 −4 Example 6 1.5 1.62.1 1.00 0.18 0.005 40 ± 5 7.5 −4 Example 7 1.2 1.8 2.1 1.00 0.16 0.00540 ± 5 7.8 −4 Comparative Example 1 5.5 4.2 2.1 1.00 0.09 0.011 40 ± 51.8 −1 Comparative Example 2 70.2 4.8 16.6 1.00 0.19 0.006 40 ± 5 5.9−10 Comparative Example 3 84.5 7.5 8.5 1.00 0.20 0.006 40 ± 5 5.8 −7Comparative Example 4 82.2 5.4 5.8 1.00 0.18 0.006 40 ± 5 5.6 −9Comparative Example 5 200 5.8 69.8 1.00 0.18 0.006 40 ± 5 5.5 −13 *¹BKNproduction is a fraction of the BKN value (1) of the non-contacted bloodused as a control. *²XPS indicates the value [N]/[S] in ComparativeExample 1 and [O]/[S] in Examples and other Comparative Examples. *³Thevalue for a porous membrane at pH 7.4.

It can be seen that bradykinin production was suppressed at a low levelin the method of Comparative Example 1 because negative charges have notbeen introduced. However, the hollow fiber of the Comparative Example 1leaked much albumin, resulting in low fractionation performance. In themethods of Comparative Examples 2-5, on the other hand, the amount ofleaked albumin was suppressed at a low level and fractionationperformance was improved due to the effect of negative charges. However,because sulfone groups having negative charges are localized on theinner surface or the blood contact surface, not only was bradykininproduction increased, but also LDH and protein adsorption wereincreased. In either case, these porous membranes could not be put intopractice.

In contrast, in the products obtained in Examples 1-7, leaking ofalbumin was considerably suppressed and fractionation performance wasimproved due to static repulsion of introduced negative charges, andbradykinin production, LDH, and protein adsorption were significantlysuppressed. These results indicate that the method of the presentinvention can excellently separate and remove low molecular weightplasma proteins and later stage glycosylated proteins that causeamyloidosis, without inducing biological reactions undesirable forliving bodies such as blood clotting, complement activity, andbradykinin production, while suppressing leakage of plasma albumin tothe minimum.

Next, examples for manufacturing an asymmetric porous membrane byintroducing a solution of negatively charged materials from oppositeside of the dense layer will be described.

Example 8

An embodiment of the membrane manufacturing method and membraneproperties of Example 8 will be described referring to FIG. 2.

As a substrate membrane, a polysulfone hollow fiber membrane with agradient asymmetric structure having a dense layer (a) inside and asupporting later (c) outside of the existing membrane, which is aultrafilter membrane in which the fractional molecular weight of thedense layer (a) is 60-100 kD, was selected. Such a membrane cannot beused in a usual blood treatment because of too large an amount ofalbumin leaked (a large sieving coefficient).

A charged layer (e) was introduced into the supporting layer immediatelybelow the dense layer of the above membrane. Specifically, a chargedpolymer solution (d) of an aqueous solution of a dilute (about 1%)proteoheparin with a high molecular weight (about 200-500 kD) wasprepared. Then, as shown in FIG. 2A, the polymer solution (d) wasreversely filtered from outside to inside of the hollow fiber membrane(in the direction of the arrow) to cause proteoheparin to be captured bythe supporting layer (c) immediately below the dense layer of themembrane as negatively charged molecules, thereby forming a chargedlayer (e) shown in FIG. 2B.

Immediately following that, a fixing solution (0.075 M sodiummetaperiodate-0.037 M lysine-pH 6.2 buffer solution) (f) was reverselyfiltered (in the direction of the arrow) in the same manner, as shown inFIG. 2C, to convert the diol group on the heparin sugar chain 5-ringinto an aldehyde group by oxidizing with sodium metaperiodate andcrosslink polysaccharide chains of different molecules by a lysinemolecule having two amino groups, whereby the polymer molecules wereentwined in the mesh structure of polysulfone polymer and immobilizedimmediately below the dense layer. Since sodium metaperiodate alsooxidizes the bisphenol ether bond of polysulfone polymer into analdehyde, this aldehyde group formed a linkage with the aldehyde groupsof the oxidized heparin via a lysine bridge. A part of heparin moleculeswere directly fixed onto the membrane via the chemical bond. Excessfixing solution was removed by sufficiently washing with water.

When heparin with a molecular weight (7-25 kD) that is smaller thanproteoheparin was used as a negatively charged substance, the heparinmolecules were preferably enlarged in advance by polymerization using acrosslinking agent, whereby the heparin molecules were effectivelycaptured by a supporting layer (a porous layer).

The charged membrane prepared in this manner was an asymmetric porousmembrane having a dense layer with a fractional molecular weight of60-100 kD and a thickness of 1 μm as the innermost layer and containingnegative charges in a supporting layer immediately below the dense layerand having a pore diameter larger than that of the dense layer(fractional molecular weight: 100 kD or more). This charged membranesatisfied the basic characteristics shown in FIG. 1.

If the asymmetric porous membrane prepared in this manner was used forhemocatharsis (hemodialysis, blood filtration, or hemodialysisfiltration), the removal rate of toxins ranging from small to largemolecular weight toxins with a molecular weight of 30-80 kD can beincreased, while suppressing loss of albumin with a molecular weight of66 kD to an allowable range (2-6 g).

Example 9

Among polyether sulfone hollow fiber membranes with a gradientasymmetric structure having a dense layer inside and a supporting lateroutside of the existing membrane, a ultrafilter membrane in which thecutt off molecular weight of the dense layer is 20-40 kD was selected.An aqueous solution of a dilute (about 1%) heparan sulfate proteoglycanwith a high molecular weight (about 100-200 kD) was prepared andreversely filtered from outside to inside the hollow fiber membrane tocause the heparan sulfate proteoglycan to be captured by the supportinglayer immediately below the dense layer of the membrane, thereby forminga charged layer. During reverse filtration, a crosslinking promoter(0.075 M sodium metaperiodate-0.037 M lysine-pH 6.2 buffer solution) wasfed from the hollow fiber membrane side and dispersed below the denselayer to initiate the polymerization reaction. This method couldimmobilize heparin immediately below the dense layer with certainty.

By filtration in the same manner, the diol group on the heparin sugarchain 5-ring was converted into an aldehyde group by oxidizing withsodium metaperiodate and polysaccharide chains of different moleculeswere crosslinked by a lysine molecule having two amino groups, wherebythe polymer molecules were entwined in the mesh structure of polysulfonepolymer and immobilized immediately below the dense layer. Since sodiummetaperiodate also oxidized the bisphenol ether bond of polyethersulfone polymer into an aldehyde, this aldehyde group formed a linkagewith the aldehyde groups of the oxidized heparin via a lysine bridge. Apart of heparan sulfate molecules were directly fixed onto the membranevia the chemical bond. Finally excessive fixing solution was removed bysufficiently washing with water.

The charged membrane prepared in this manner was an asymmetric porousmembrane having a dense layer with a cutt off molecular weight of 20-40kD and a thickness of 1 μm as the innermost layer and containingnegative charges in a supporting layer immediately below the dense layerand having a pore diameter (cutt off molecular weight: 40 kD or more)larger than that of the dense layer. This charged membrane satisfied thebasic characteristics shown in FIG. 1.

If urine, for example, is filtered using the asymmetric porous membranethus prepared, α1-microglobulin, a protein with a molecular weight of 33kD contained in a large amount in urine, and β2-microglobulin, an acidicprotein with a molecular weight of 11.7 kD, can be blocked at the sametime, and peptides and proteins with a molecular weight of 20 kD or lesscontained in urine in a very small amount can be separated in thefiltrate at a high yield.

The effect of the membrane having a dense layer with a cutt offmolecular weight of about 60-80 kD and a thickness of 1 μm and anegatively charged layer immediately below the dense layer with a cuttoff molecular weight of about 80 to several hundred kD in blood with anobjective of curing diseases such as renal failure or hepatic failure isshown below. Hemofiltration, hemodiafiltration, and hemodialysis can beselected as the blood purification therapy. The hemofiltration andhemodialysis filtration are more effective.

Since the separating membrane of the present invention has a large poresize in the dense layer as compared with conventional blood purifyingmembranes, removal performance of large toxic molecules with a molecularweight of 20 kD or more that had been difficult to remove using aconventional treatment has been significantly improved in the presentinvention.

FIG. 3A shows the results of experiment to determine the sievingcoefficient (a membrane permeation index determined by dividing theconcentration of a solute in a filtrate by the concentration of thesolute in a loaded liquid) for α1-microglobulin with a molecular weightof 33 kD when human plasma was filtered. The following three filtersamples were used.

(1) A blood filter membrane having the highest performance amongcommercially available conventional blood filter membranes (common bloodfilter membrane). The material is a polysulfone membrane with a cut offmolecular weight of 10-20 kD.

(2) A large pore size ultrafilter membrane with an asymmetric porousstructure, not used for blood filtration. The material is a polyethersulfone membrane with a cut off molecular weight of 60-80 kD. and

(3) A large pore size ultrafilter membrane with an asymmetric porousstructure, with a negatively charged layer introduced (large pore sizecharged membrane).

The α1-microglobulin sieving coefficient was naturally high in thelatter two large pore size membranes. These two membranes possessedalmost the same sieving coefficient value, indicating that the sievingfunction is not affected by introducing a negatively charged substance.

On the other hand, although the dense layer of the large pore sizeultrafilter membrane with an asymmetric porous structure allows albuminmolecules to permeate therethrough, the albumin blocking performance canbe optimally controlled due to electrical repulsion of albumin moleculesby the negatively charged layer immediately below the dense layer. FIG.3B shows the results of the albumin sieving coefficient determination ofthe above three membranes. As can be seen from the Figure, the largepore size charged membrane (3) with the same large pore size structureas that of the large pore size membrane (2) suppressed albuminpermeation to a level equivalent to or lower than the common bloodfilter membrane due to introduction of negative charges. As a result,performance of separating macromolecular toxins (for example,α1-microglobulin) that is the subject to be removed but can be removedonly with difficulty by the molecular size difference from albumin thatis the subject to be blocked has remarkably increased.

INDUSTRIAL APPLICABILITY

The asymmetric porous membrane of the present invention can separate aspecific solute and/or dispersoid from a multicomponent solution. Themembrane has a double barrier structure, one a size barrier and theother a charge barrier of negative charges. In addition, negativecharges are present inside the membrane, with at least the outermostsurface of the dense layer being substantially free from electriccharges.

As a result, the asymmetric porous membrane of the present inventionexhibits remarkably improved performance to separate a solute and/ordispersoid from a multicomponent solution, while preventing a solutionto be processed from chemically or biologically reacting due to electriccharges.

The asymmetric porous membrane of the present invention can be usedparticularly preferably when the liquid to be processed is blood and canexcellently separate and remove low molecular weight plasma proteins andlater stage glycosylated proteins that cause amyloidosis, withoutinducing biological reactions undesirable for living bodies such asblood clotting, complement activity, and bradykinin production, whilesuppressing leakage of plasma albumin to the minimum.

1. A method for manufacturing an asymmetric porous membrane, comprisingproviding a porous substrate membrane with an asymmetric structuremainly made from a synthetic polymer substantially free from electriccharges and having a dense layer on the side on which a liquid is loadedand filtering or diffusing a solution of a negatively charged polymerthat can be blocked by the dense layer from the side opposite to thedense layer to block the negatively charged polymer from permeatingthrough the dense layer, thereby introducing negative charges to thepart excluding the dense layer and immobilizing the negatively chargedmaterial to the part excluding the dense layer.
 2. The method accordingto claim 1, wherein the negative charges are introduced at a highdensity immediately below the dense layer by blocking the negativelycharged polymer immediately below the dense layer.